NDT Advance Access originally published online on May 25, 2006
Nephrology Dialysis Transplantation 2006 21(9):2380-2390; doi:10.1093/ndt/gfl243
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Original Articles: Experimental Nephrology
Role of integrin-linked kinase in epithelial–mesenchymal transition in crescent formation of experimental glomerulonephritis
1 Department of Pediatrics, The Institute of Health Bioscience, The University of Tokushima Graduate School, Tokushima and 2 Department of Molecular Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan
Correspondence and offprint requests to: Shoji Kagami, MD, PhD, Department of Pediatrics, The Institute of Health Bioscience, The University of Tokushima Graduate School, Kuramoto-cho-3-chome, Tokushima 770-8503, Japan. Email: kagami{at}clin.med.tokushima-u.ac.jp
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Abstract |
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Background. Glomerular parietal epithelial–mesenchymal transition (EMT) is a key event in crescent formation of glomerulonephritis (GN). Integrin-linked kinase (ILK) is an integrin cytoplasmic-binding protein that has been implicated in the regulation of cell adhesion, extracellular matrix organization and EMT. Transforming growth factor-ß (TGF-ß) is involved in the induction and progression of EMT in several tissues.
Methods. To investigate whether ILK is involved in the crescent formation in GN, we studied the expression of ILK protein and activity in crescentic GN induced in Wistar Kyoto (WKY) rats. In addition, we investigated whether transforming growth factor-ß1 (TGF-ß1) could induce glomerular EMT and ILK by using cultured parietal epithelial cell (PEC).
Results. The expression of ILK was strongly induced in cellular crescents at day 7 and followed by a decrease in fibrocellular crescents at day 28. ILK-expressing cells in cellular crescents were double-positive for protein gene product 9.5 (PEC marker), 
-smooth muscle actin (-SMA, myofibroblasts marker) and TGF-ß1, indicating a possible contribution of ILK and TGF-ß1 to EMT in crescent formation in GN. Consistent with the finding of histological ILK expression in crescents, western blot and kinase activity assay showed an increase in both ILK protein and activity, peaking at day 7 of GN (3.7- and 3.5-fold of control, respectively). The expression of ILK increased to 3.1-fold of control when EMT was induced in cultured PEC by TGF-ß1.
Conclusion. The present results provide the first evidence that expression and activity of ILK are increased in cellular crescents of experimental GN. Enhanced expression and activity of ILK, possibly by TGF-ß1, is associated with the induction of EMT by PEC and thereby, may participate in the formation of cellular crescents in GN.
Keywords: crescent formation; epithelial–mesenchymal transition; integrin-linked kinase
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Introduction |
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Integrin-linked kinase (ILK) is a serine/threonine kinase which interacts with cytoplasmic domains of ß1 integrins (ß1-IG) and has been implicated in the regulation of cell adhesion, proliferation and extracellular matrix (ECM) organization [1–3]. ILK is activated by ß1-IG-mediated adhesion to ECM or stimulation with growth factors such as transforming growth factor-ß (TGF-ß). ILK is considered to function as the effector of PI3-K signalling, which regulates protein kinase B (PKB)/Akt and glycogen synthase kinase-3 (GSK-3) activity and thereby mediates a wide range of cellular processes [1,4]. Recently, it has been suggested that ILK participates in epithelial–mesenchymal transition (EMT) involved in embryonic development, oncogenesis, metastasis of malignant cells and fibrosis of organs including the kidney [1,5,6].
EMT is a phenomenon characterized in epithelial cells by the loss of epithelial markers and acquisition of a mesenchymal phenotype, which is recognized by the expression of -smooth muscle actin (-SMA) [5]. It is induced by cytokines and growth factors, in which TGF-ß plays a central role in mediating the EMT [1,5]. Through the EMT, an epithelial cell is differentiated into a myofibroblast, which is spindle shaped and expresses -SMA [7,8]. Many studies have revealed that interstitial fibroblasts originating from epithelial cells through EMT play a key role in renal fibrosis in various kidney diseases [5,7,9–12]. Subsequently, Li et al. showed that ILK is a critical mediator for renal tubular EMT and thereby contributes to the development of renal fibrosis [5].
EMT is also suggested to be involved in the organization of glomerular crescents in human and rat glomerulonephritis (GN) [13,14]. Glomerular crescent formation is a prominent cell biological feature of rapidly progressive GN and is associated with a poor prognosis. There are three distinct stages of glomerular crescent progression. Initially cellular crescents are formed and they develop into fibrous crescents through the fibrocellular crescents in chronic renal injury. It is known that cellular crescents consist of macrophages, parietal epithelial cells (PEC) of Bowman's capsule and -SMA-positive myofibroblasts [13,14]. Notably, two recent reports indicated that -SMA-positive myofibroblasts within the crescents are derived from PEC via EMT [7,8]. However, the detailed mechanisms of PEC in undergoing transdifferentiation into -SMA-positive myofibroblasts and its participation in the formation of glomerular crescents remains unclear.
Therefore, we studied the expression of protein gene product 9.5 (PGP 9.5, PEC marker), -SMA, ILK and TGF-ß1, and ILK activity in anti-glomerular basement membrane (GBM) serum-induced crescentic GN of rats, in order to elucidate the possible contribution of ILK to the EMT and crescent formation in GN. Additionally, we investigated whether PEC could undergo EMT in association with ILK induction after TGF-ß1 stimulation.
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Methods |
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Anti-GBM GN in WKY rats
Progressive anti-GBM GN was induced in 7-week-old male WKY rats (Charles River Japan Inc., Kanagawa, Japan) by a single intravenous injection of rabbit anti-rat GBM antiserum (0.2 mg/100 g body weight) via the tail vein. Groups of six rats were sacrificed on days 3, 7 and 28 after the injection for histological examination and isolation of glomeruli. A group of six normal age-matched, unmanipulated rats was used as a control. Rat glomeruli were purified by the graded sieving method [15], homogenized and lysed for 30 min on ice in a lysis buffer (Cell Signaling Technology, Inc., Beverly, MA, USA). The supernatants were collected after centrifugation at 15 000g at 4°C for 20 min and used for biochemical examination.
Kidney function studies
The urine of all rats was obtained in a 24 h collection by using metabolic cages (TECNIPLAST, Buguggiate, Italy). The amount of protein excreted into the urine was measured by Bradford method using Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, CA, USA). Blood samples were obtained from all the rats at the time of sacrifice. Serum blood urea nitrogen (BUN) levels were measured by the urease–ultraviolet method using Urea NB (Wako Pure Chemical Industries, Ltd., Osaka, Japan).
Histological examination
Kidney tissues were fixed in 10% formalin and embedded in paraffin. Paraffin sections of 3 µm were stained with periodic acid–Schiff reagent (PAS). Glomeruli were considered to exhibit crescent formation when at least two layers of cells were observed in the Bowman's space. The numbers of crescents were counted in 100 glomeruli per rat and expressed as the mean percentage for each group.
Paraffin-embedded kidney tissues were sectioned, deparaffinized and incubated with either anti-PGP 9.5 monoclonal antibody (mAb) (13C4, Biogenesis, Poole, England) as a PEC marker, anti--SMA mAb (1A4, Sigma-Aldrich Co., St Louis, USA) as a myofibroblast marker, anti-ED1 (specific rat macrophage marker) mAb (MCA341R, Serotech, Oxford, UK) as a macrophage marker or anti-ILK mAb (65.1.9, Upstate, NY, USA), respectively. Tissues were then stained by standard immunoperoxidase procedures according to the manufacturer's instructions. The total number of nucleated cells and the number of PGP 9.5, -SMA, ED1 and ILK-positive cells within the glomerular crescents were counted. The mean number of cells was calculated from at least 20 crescents per rat and expressed as the percentage of positive cells in each group.
Double immunostaining was performed using 3 µm frozen sections of kidney tissues. To detect the involvement of EMT in crescent formation, the sections were incubated with rabbit anti-PGP 9.5 antibody (AFFINITI Research Product Ltd., Exeter, UK) and anti--SMA mAb (1A4) followed by fluorescein (FITC)-labelled donkey anti-rabbit IgG antibody and rhodamine (TRITC)-labelled donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). For characterization of ILK-positive cells within crescents, sections stained with goat anti-ILK antibody (Santa Cruz, CA, USA) and FITC-labelled donkey anti-goat IgG antibody (Jackson ImmunoResearch Laboratories Inc.) were further incubated with either anti-PGP 9.5 mAb (13C4), anti--SMA mAb (1A4), anti-ED1 mAb (MCA341R) or anti-synaptopodin mAb (G1D4, PROGEN Biotechnik, Heidelberg, Germany), followed by TRITC-labelled donkey anti-mouse IgG antibody.
To examine the expression of TGF-ß1 and its well-known target molecules, ß1-IG and fibronectin (FN) in nephritic glomeruli, the sections were incubated with either goat anti-TGF-ß1 antibody (Santa Cruz, CA, USA), rabbit anti-ß1-IG subunit antibody or rabbit anti-FN antibody (Chemicon International, Temecula, CA), followed by FITC-labelled anti-goat IgG antibody or FITC-labeled donkey anti-rabbit IgG antibody, appropriately. For semiquantitative evaluation of the expression of TGF-ß1, ß1-IG and FN in glomerular crescents, sections were graded based on the extent of positive crescent area as follows: (–) negative; (±) weak; (+) mild; (++) moderate and (+++) strong. To further define TGF-ß1-positive cells within crescents, the sections stained with goat anti-TGF-ß1 antibody and FITC-labelled donkey anti-goat IgG antibody were incubated with either anti-PGP 9.5 (13C4), anti--SMA mAb (1A4), anti-ED1 mAb (MCA341R), anti-synaptopodin mAb (G1D4) or anti-ILK mAb (65.1.9), followed by TRITC-labelled donkey anti-mouse IgG antibody.
Cell culture
Rat PEC line was established and characterized as previously described [13,16]. PEC line was cultured with K1-3T3 medium with some modifications. About 50–60% confluent PEC was incubated in serum-free Dulbecco's modified eagle's medium (DMEM) (Gibco BRL, Grand Island, NY, USA) for 48 h to achieve PEC quiescence and then treated with 10 ng/ml recombinant TGF-ß1 (R&D Systems, Minneapolis, MN, USA) for 12, 24 and 48 h. Cells were rinsed with cold phosphaste-buffered saline (PBS) and harvested for lysis with an ice-cold lysis buffer (Cell signaling Technology, Inc., Beverly, MA, USA). The supernatants were collected after centrifugation at 15 000 g at 4°C for 20 min. As a time-matched control, PEC lysate was obtained from cell culture without TGF-ß1 stimulation. Furthermore, morphological changes were detected by phase-contrast microscopy to assess whether TGF-ß1 could induce characteristic EMT in PEC.
Western blot analysis
Protein samples (30 µg) were separated by 12.5% SDS–polyacrylamide gels (PAGE) and transferred to nitrocellulose membranes (Amersham Bioscience, Piscataway, NJ, USA). The membranes were probed with either an anti-ILK mAb (65.1.9), anti--SMA mAb (1A4), rabbit anti-ß1–IG subunit antibody or rabbit anti-FN antibody and then incubated with a horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Cell Signaling Technology, Inc., Beverly, USA) or an HRP-conjugated donkey anti-rabbit IgG (Cell Signaling Technology, Inc., Beverly, USA), respectively. Electrophoresis for ß1-IG was only performed under non-reduced condition, as previously described [17]. Immunoreactive proteins were detected with an enhanced chemiluminescence detection system (Amersham Corp., Arlington Heights, IL). Blots were stripped and probed with an anti-ß-actin mAb (AC-15, Sigma-Aldrich, St Louis, MO) to confirm equal loading and transfer. Bands were quantitated by ImageJ 1.33u (National Institute of Health, USA) and the fold expression was indicated as the relative protein level.
ILK activity
ILK activity was determined in cell lysates by immune complex kinase assay as previously described with some modifications [18]. Pre-cleared cell lysates (100 ug) were incubated with anti-ILK mAb (65.1.9) and protein G beads overnight at 4°C. Immune complexes were washed twice with a lysis buffer followed by two washes with kinase buffer (Cell signaling Technology, Inc., Beverly, USA). Washed pellets were incubated with kinase buffer supplemented with 200 µM adenosine triphosphate (ATP) and 250 ng of the commercially available Akt/PKBa (unactivate) (Upstate Biotechnology, NY, USA) for 60 min at 30°C. The kinase reaction was terminated by the addition of SDS sample buffer and the supernatants were boiled for 5 min at 100°C and resolved by SDS/PAGE (12.5% gels). Membranes were probed with phospho-Akt (ser473) mAb (587F11, Cell Signaling Technology, Inc., Beverly, USA) followed by incubation with an HRP-conjugated anti-mouse antibody. Immunoreactive protein was detected with an enhanced chemiluminescence detection system (Amersham Corp., Arlington Heights, IL, USA). Blots were stripped and reprobed with a rabbit anti-total Akt antibody (Cell Signaling Technology, Inc., Beverly, USA) to confirm equal loading and transfer. Bands were quantitated by ImageJ 1.33 u (National Institute of Health, USA) and the fold expression was indicated as relative ILK activity level.
Statistical analysis
Western blot analysis and ILK activity assay were performed at least three times and the representative results were shown as figures. Results were presented as mean ± SD. Statistical analysis of the data was performed by t-test. Values of P < 0.05 were considered to be statistically significant.
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Renal histology, function and proteinuria
At day 3 after the disease induction, glomeruli showed severe endocapillary proliferation with focal necrotizing and mesangiolytic damage. Small crescents in only 10.7 ± 4.5% of nephritic glomeruli were observed. At day 7 of GN, marked cellular crescent formation was observed in 82.0 ± 6.6% of glomeruli as a result of severe glomerular damage. Thereafter, cellular crescents and proliferative glomerular lesions progressed to fibrocellular crescents with reduced cellularity and sclerotic ones at day 28 of GN, respectively (Figure 1). A significant increase in urinary protein was detected from day 3 (17.0 ±2.9 mg/day) and increased progressively at day 7 (153.7 ± 23.4 mg/day), day 14 (265.0 ± 41.2 mg/day), day 21 (322.5 ± 53.4 mg/day) and day 28 (306.1 ±42.1 mg/day) of GN. The level of serum BUN significantly increased after day 7 of GN (26.86 ±1.06 mg/dl) and continued to increase until the end of the experiment (47.19 ± 5.72 mg/dl).
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Cell components in glomerular crescents of GN
Immunohistochemical study using specific markers revealed the cell types, which consisted of crescents and their kinetics during the course of GN. Crescents comprised various proportions of ED1, PGP 9.5,
-SMA and ILK-positive cells, depending on the stage of glomerular crescents (Figures 2 and 3). The percentage of ED1-positive macrophages in crescents peaked at day 3 (55.0 ± 22.0%), the early phase of crescentic GN, and declined markedly afterwards (Figures 2A and 3A–D). PGP 9.5-positive cells having a phenotype of PEC were partially induced at day 3 of GN (25.1 ± 11.1%). They became the most abundant cellular crescents at day 7 (66.1 ± 14.5%) and decreased in fibrocellular crescent at day 28 of GN (Figures 2B and 3E–H). In contrast to the kinetics of PGP 9.5-positive cells, the number of -SMA (a phenotype marker of myofibroblast)-positive cells rapidly increased at day 7 (57.2 ± 20.0%) and continued to increase over 98% in fibrocellular crescents at day 28 of GN (98.4 ± 1.2%) (Figures 2C and 3I–L). ILK was primarily expressed in the glomerular podocytes of control rat glomeruli. It was also weakly positive in mesangial cells, endothelial cells and PEC. Prior to the induction of -SMA in nephritic glomeruli, ILK was detected early, at day 3 (23.3 ± 20.4%), then strongly induced in crescentic cells at day 7 (68.7 ± 24.2%), followed by a decrease in fibrocellular crescents at day 28 of GN (23.2 ± 14.6%) (Figures 2D and 3M–P). The transition of ILK-positive cells is closely parallel to that of PGP 9.5-positive cells in the stages of glomerular crescents, strongly suggesting that ILK-positive crescentic cells are derived from proliferating PEC after glomerular injury. The synaptopodin-positive cells (glomerular podocytes) were not seen in crescents at any time point of GN (data not shown).
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Involvement of ILK in glomerular EMT of crescentic GN
To confirm the involvement of EMT in crescent formation of GN, double staining of nephritic glomeruli with anti-PGP 9.5 mAb and anti-
-SMA mAb was performed. Abundances of double-positive cells were observed within the cellular crescent at day 7 of GN (Figure 4A–C), indicating that PEC aggressively acquires a phenotypic marker of myofibroblast by a process of EMT in cellular crescent formation. Furthermore, a possible role of ILK in the glomerular EMT was also examined by double staining (Figure 4D–F). Most of the ILK-expressing cells were clearly double-positive for PGP 9.5 (Figure 4D) and -SMA (Figure 4E) but not for ED1 (Figure 4F) within the cellular crescent at day 7 of GN. Thus, taken together with the results of the time course of glomerular crescent-forming cells, ILK induction appears to precede the EMT by PEC and may contribute to the cellular crescent formation in this model of GN. In the control double staining of the normal rat glomeruli, PGP 9.5 was detected for PEC on the Bowman's capsule and -SMA was not seen within the glomeruli (Figure 4G). Moreover, ILK-expressing glomerular cells were all negative for PGP 9.5, -SMA and ED1 under normal conditions (Figure 4H–J).
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The expression of TGF-ß1, ß1-IG and FN in crescentic GN
To gain insights into the mechanism underlying glomerular EMT in GN, the expression of TGF-ß1, ß1-IG and FN in glomerular crescents was investigated (Figure 5). Faint staining of TGF-ß1 was detected in normal rat glomeruli (Figure 5A). Following the glomerular injury, segmental mesangial staining of TGF-ß1 was observed at day 3 of GN (Figure 5B). TGF-ß1 was strongly induced in cellular crescents at day 7 (Figure 5C), and steadily increased in fibrocellular crescents at day 28 of GN (Figure 5D). The expression of ß1-IG was strongly induced in cellular crescents at day 7 and then decreased in fibrocellular ones at day 28 of GN, showing a parallel transition with the expression of ILK to which ß1-IG binds (Figure 5E–H). On the other hand, FN, one of the ß1-IG-ligand ECM components, appeared at day 7, and became more prominent at day 28 of GN (Figure 5I–L). The level of FN expression paralleled that of
-SMA-positive cells within crescents. The transitions of TGF-ß1, ß1-IG and FN are summarized in Table 1. Double-staining revealed that TGF-ß1-expressing cells within cellular crescents at day 7 of GN were positive for PGP 9.5 or -SMA and ILK (Figure 6A–C), but negative for ED1 or synaptopodin (Figure 6D and E), indicating that TGF-ß1-expressing cells acquire a PEC myofibroblast phenotype that is a main composer of cellular crescents in GN.
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ILK protein expression and activity in nephritic glomeruli
Western blot analysis revealed that ILK protein and activity were induced in rats with anti-GBM GN in a time-dependent fashion (Figure 7). A significant increase of ILK protein and activity were detected as early as day 3 after glomerular injury, peaked at day 7 (3.7- and 4.0-fold of control level, respectively) and then declined at day 28 of GN (Figure 7A, C, E and F). Following the increase of ILK protein and activity at day 3, a rapid induction of
-SMA protein was seen at day 7 of GN. Then, -SMA protein progressively increased at day 28 of GN (Figure 7B and D). These results regarding ILK and -SMA expression were consistent with those of histological expression of each protein during that course of glomerular crescent formation.
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P < 0.01 vs day 3; 
P < 0.01 vs day 7. Values are mean ± SD. The results shown were obtained from a representative experiment of at least three independent experiments.TGF-ß1 induces ILK and EMT by cultured PEC
Finally, the potential role of TGF-ß1 and ILK in the induction of EMT by PEC seen in crescentic GN was investigated using a cell culture system. As shown in Figure 8, TGF-ß1 treatment for 48 h induced fibroblastic morphology (a characteristic feature of EMT) in PEC (Figure 8B), which originally showed a cobblestone epithelial morphology (Figure 8A). Furthermore, western blotting revealed that TGF-ß1 also significantly increased the expression of ILK and
-SMA by PEC in a time-dependent manner (Figure 9). ILK began to increase significantly after 24 h treatment (2.4-fold of control) and continued to increase until the end of observation (3.1-fold of control) (Figure 9A and E). Though -SMA was not observed in control culture condition, it started to appear after a 24 h incubation with TGF-ß1 and reached the peak at 48 h after treatment (3.0-fold of 12 h treatment) (Figure 9B and F). These results suggested that TGF-ß1 is a potential inducer of ILK and EMT and that enhancement of ILK expression is associated with induction of EMT by PEC in vivo. In addition, TGF-ß1 significantly increased the expression of ß1-IG and FN, well-known target molecules for TGF-ß1, by cultured PEC. ß1-IG began to increase significantly after a 12 h treatment (1.4-fold of control) and continued to increase to 2.3- and 4.2-fold of control at 24 h and 48 h of treatment, respectively (Figure 9G and K). In contrast, FN was increased early to 2.0-fold of control after a 12 h treatment and was maintained at a significantly increased level in cultured PEC (Figure 9I and L). Thus, TGF-ß1 clearly increased the PEC expression of ILK and -SMA as well as ß1-IG and FN, all molecules strongly induced in glomerular cellular crescents at day 7 of GN. The expression of ILK, -SMA, ß1-IG and FN in control culture without TGF-ß1 was not significantly changed at time points examined (Figure 9B, D, H and J, respectively).
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Discussion |
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EMT is a dynamic process in which epithelial cells lose their characteristics and acquire the mesenchymal phenotype (
-SMA-positive myofibroblasts) [7,19,20]. The transformed cells express -SMA and possess the increased ability to proliferate, migrate and synthesize a massive amount of extracellular matrix while they lose epithelial cell phenotype. EMT is necessary for embryonic development, tumour progression and organ fibrosis [21,22]. In the present study, we provide the first evidence that ILK is strongly expressed and activated in cellular crescents and show the possible contribution of ILK to the induction of EMT by PEC and to the formation of glomerular cellular crescents in a progressive model of rat GN. This conclusion is supported by the following results: Immunofluorescence study showed that ILK and PGP 9.5, a marker of PEC, were weakly induced in early crescentic cells at day 3 of GN. Then, both were strongly expressed in cellular crescents at day 7 of GN, which simultaneously expressed -SMA, indicating that most ILK-positive crescent cells were PEC acquiring myofibroblast phenotype in the transitional phase of dynamic phenomenon of EMT. Later, the number ILK- and PGP 9.5-positive cells was decreased in the fibrocellular crescent at day 28 of GN while -SMA-positive myofibroblasts were dominant. Although the origin of -SMA-positive myofibroblasts of the late stage remains uncertain, some of them may be derived by transdifferentiation of PEC, others may arise from migration of periglomerular myofibroblasts into Bowman's space through the ruputured Bowman's basement membrane evident in fibrocellular crescents [23]. Similarly, Fujigaki et al. [24] also suggested, using immunohistochemical methods, that the origin of the -SMA-positive cells in the cellular crescents in this model of GN is PEC, whereas that of -SMA-positive cells in the fibrocellular crescents may be transdifferentiated PEC or invaded periglomerular myofibroblasts. Recently, using transgenic mice expressing ß-galactosidase specifically in their podocytes, Moeller et al. [25] showed the possible contribution of podocytes to early crescent formation in GN [25]. Interestingly, they also found that crescentic cells derived from podocytes did not express any podocyte-specific markers such as Wilm's tumor-associated gene (WT-1), synaptopodin, anti-glomerular epithelial protein (GLEPP-1) and podocin. Accordingly, it is possible that ILK-positive and PGP 9.5-negative cells within cellular crescents might be derived in part from glomerular podocytes.
In parallel with the degree of histological ILK expression in the glomerular crescent, the levels of ILK protein and activity of isolated nephritic glomeruli peaked at day 7 and then decreased at day 28 of GN, when expression of -SMA was still high. There is a similar line of study that the peak of ILK expression at day 7 precedes that of -SMA expression at day 14 in a model of renal interstitial fibrosis induced by unilateral ureteral obstruction [5,26]. In this model, tubular induction of ILK has been shown to be accompanied by a phenotypic conversion of tubular epithelial cells to -SMA-positive cells by a process of EMT. The transdifferentiated cells lose the epithelial phenotype through EMT and can invade interstitial compartments, thereby participating in interstitial fibrosis [27–29]. Thus, ILK expression and activity are likely involved in the acquisition of the mesenchymal phenotype and the early event in EMT necessary for the development of tissue fibrosis.
Accumulating evidences from many cell biological studies indicated that ILK is a critical mediator for induction of EMT. Overexpression of ILK in two epithelial cell lines (intestinal epithelial cells (IEC)-18 intestinal epithelial cells and scp2 mammary epithelial cells) with wild-type ILK cDNA vector has been observed to induce EMT accompanied with down-regulation of E-cadherin (one of the caretakers of epithelial phenotype), up-regulation of FN assembly, and invasive phenotype [4,30]. Forced expression of wild-type ILK cDNA in renal tubular epithelial cells also induces numerous key events of EMT, including loss of epithelial E-cadherin, induction of FN expression and enhanced cell proliferation, migration and invasion [5]. Taken together, these results support the inference that ILK is an endogenous strong inducer for EMT [1,31].
It is now known that EMT is regulated by multiple regulators such as growth factors, cytokines, hormone, adhesion molecules, ECM and intracellular signalling molecules including ILK [26,32–35]. Especially, TGF-ß is an sole factor that can initiate and complete the EMT course. Recently, TGF-ß1 has been shown to enhance the expression of ILK in many cell types [5,31,36]. Indeed, the increased expression of TGF-ß1 is observed when ILK and -SMA are strongly induced in cellular crescents of this GN model. Furthermore, our in vitro study showed that TGF-ß1 could induce not only the expression of ILK, but also the de novo expression of -SMA in cultured PEC. Consistent with this -SMA induction, PEC showing a cobblestone epithelial morphology presented a fibroblastic morphology (a characteristic feature of EMT) after TGF-ß stimulation. In addition, treatment of PEC with TGF-ß1 also increased the expression of ß1-IG and FN which are strongly induced in cellular crescents [8]. In turn, each function of ß1-IG and FN has been shown to be involved in TGF-ß-induced EMT progression [37,38]. Taken together, TGF-ß1 and its controlled molecules, such as ILK, -SMA, ß1-IG and FN seem likely to work in coordination for the induction of EMT by PEC. It is therefore possible to speculate the following scenario for the formation of glomerular crescents in this model of GN: (i) Following the injection of rabbit-GBM serum into WKY rats, diffuse endocapillary proliferative GN developed at day 3 of GN. Nephritic glomeruli had increased the expression of TGF-ß1. (ii) Secreted TGF-ß1 along with various inflammatory molecules into Bowman's capsule stimulates the expression and activity of ILK by PEC. (iii) PEC acquires the mesenchymal phenotype and begins to express -SMA and shows the enhanced ability to proliferate to form glomerular cellular crescents, accompanied by enhanced expression of ß1-IG and FN expression (day 7 of GN). (iv) Finally, transformed cells (-SMA positive myofibroblasts) lost the epithelial marker (PGP 9.5) and secreted a large amount of extracellular matrix proteins and progressed to fibrocellular crescents (day 28 of GN).
Regarding the involvement of TGF-ß in formation of glomerular crescents in this model of GN, Zhou et al. [39] recently provided clear evidence that TGF-ß plays a pivotal role in the development of glomerular crescents composed of -SMA positive myofibroblasts. They showed that the treatment of the same GN of rats with adenovirus vector (Tß-ExR), secreted a soluble TGF-ß type II receptor, significantly ameliorated the -SMA positive crescentic cells and resulted in a decrease of crescent formation, suggesting a critical role of TGF-ß in transition of glomerular PEC into myofibroblasts within glomerular crescents. A similar approach inhibiting TGF-ß action in this type of GN may provide in vivo data indicating the role of ILK in -SMA induction and EMT progression during the course of glomerular crescent formation.
In the view of clinical application, TGF-ß1 antagonists such as neutralizing antibody, anti-sense oligonucleotide, decorin and Tß-ExR, seem to serve as potential inhibitors for preventing the development of glomerular crescents in GN. However, long-term suppression of TGF-ß1 action might induce severe side effects, such as the wasting syndrome accompanied by massive multifocal inflammation affecting multiple organs [40]. Thus, the development of potential inhibitors capable of suppressing ILK activity under in vivo conditions will provide new information regarding the substantial role of ILK in EMT that occurs in glomerular crescent formation and might lead to the discovery of a new drug candidate for the treatment of severe crescentic GN in humans.
In summary, this study showed, for the first time, that enhanced expression and activity of ILK are associated with the induction of EMT by PEC and thereby, may participate in the formation of glomerular cellular crescents in GN. Considering the potential contribution of ILK to EMT for the development of renal interstitial fibrosis the discovery of a small molecule inhibiting ILK activity in vivo will provide a new potential therapeutic method for preventing the progression of GN characterized with crescent formation.
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Acknowledgments |
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This work was supported by grants from the Japanese Ministry of Welfare (14570748, 16591035). We would like to thank Dr K. Loster and Dr W. Reutter (Freie Universitat Berlin, Germany) for providing the rabbit anti-ß1 integrin antibody. We also thank Naomi Okamoto for her excellent technical assistance. Parts of this work were published in abstract form at the 36th Annual Meeting of the American Society of Nephrology, 12–17 November 2003.
Conflicts of interest statement. None declared.
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[Abstract/Free Full Text]
Accepted in revised form: 5. 4.06
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