The results presented in this paper implicate ILK as a crucial mediator of mesenchymal transformation from tubular epithelial cells in the pathogenesis of renal interstitial fibrosis. This conclusion is supported by several lines of observation. First, endogenous ILK expression in tubular epithelial cells is induced by TGF-β1, a profibrotic cytokine that has been shown to initiate tubular EMT in vitro (2
). Second, forced expression of exogenous ILK induces numerous key events including loss of the epithelial cell-cell adhesion molecule E-cadherin, induction of fibronectin expression and its extracellular assembly, induced MMP-2 expression and secretion, and enhanced cell migration and invasion. This virtually recapitulates the major events during the entire course of tubular EMT induced by TGF-β1 (2
), underscoring that ILK is perhaps sufficient for mediating tubular EMT. Third, ectopic expression of a dominant-negative, kinase-dead form of ILK largely abrogates TGF-β1–initiated E-cadherin suppression and fibronectin induction and assembly (Figure ), corroborating the notion that ILK signaling is necessary for mediating TGF-β1’s action in tubular EMT. Fourth, ILK induction is specifically confined to renal tubular epithelium and coincides with tubular EMT in two models of chronic renal fibrosis induced by either obstructive insult or diabetic injury in mice, indicating a spatial and temporal association between ILK and tubular EMT in vivo. Finally, inhibition of ILK induction by HGF blocks TGF-β1–initiated tubular EMT in vitro and attenuates renal interstitial fibrosis in vivo. Collectively, these results present convincing arguments supporting a critical role for ILK in mediating tubular EMT. Our findings shed new light on identifying the molecular mediator and elucidating the mechanism underlying tubular EMT in the pathogenesis of chronic renal fibrosis.
Previous studies have demonstrated that tubular EMT is an orchestrated process that is regulated by a coordinated alteration in gene expression (2
). It is conceivable that the completion of the tubular EMT process depends on proper interaction of tubular cells with ECM via integrin-mediated signaling. In light of its central position in the crossroads connecting the integrins and the actin cytoskeleton (17
), ILK was hypothesized to be a candidate signaling molecule that plays a role in mediating TGF-β1–initiated tubular EMT. In this study, we have clearly demonstrated that ILK is a downstream effector of TGF-β1, because ILK is both sufficient and necessary for mediating tubular EMT (Figure through Figure ), and because its expression in tubular epithelial cells is tightly controlled by TGF-β1 (Figure ). In accordance with this, it has been demonstrated that TGF-β1 induces ILK expression in human HT-144 melanoma cell lines (33
). Of interest, despite the fact that TGF-β1 is capable of activating several signal transduction pathways (27
), ILK induction by TGF-β1 is clearly dependent upon intact Smad signaling in tubular epithelial cells, since overexpression of inhibitory Smad-7 abolishes Smad-2 phosphorylation and ILK induction (Figure ). Consistently, overexpression of Smad-7 also blocks tubular EMT induced by TGF-β1 (36
). Of note, although RhoA has been implicated in EMT (37
), TGF-β1–induced RhoA activation in tubular epithelial cells is neither dependent on Smad signaling nor on ILK (Y. Li et al., unpublished data). This suggests that Smad/ILK and RhoA are two parallel signaling pathways initiated by TGF-β1. Given the fact that RhoA is important only in TGF-β–induced stress fiber formation but not in the disruption of adherens and tight junctions (38
), it is reasonable to assume that ILK acts as a major intermediate signaling molecule that couples TGF-β1/Smad signaling and tubular EMT.
Through its interactions using distinct domains, ILK strategically bridges the integrins and actin cytoskeleton–associated proteins including PINCH, CH-ILKBP, and paxillin and transmits signal exchanges between the intracellular and extracellular compartments (17
). In addition, ILK also couples integrins and growth factor receptors to downstream signaling components (17
). Among the many functions of ILK in diverse cellular processes, perhaps the chief one is to modulate cell adhesions (20
). Accordingly, a hallmark of ILK overexpression in epithelial cells is the loss of epithelial cell-cell adhesion by downregulation of E-cadherin expression (Figure ) (20
). This is in agreement with our previous observation that suppression of E-cadherin expression is a key step that precedes other major events during tubular EMT induced by TGF-β1 (2
). Therefore, ectopic expression of ILK in tubular epithelial cells underlines that ILK signaling per se is sufficient for mediating TGF-β1’s action to initiate mesenchymal transformation of tubular epithelial cells. Because E-cadherin is an epithelial adhesion receptor that plays a pivotal role in the maintenance of the structural and functional integrity of tubular epithelium (43
), downregulation of E-cadherin will presumably lead to destabilization of epithelial sheet integrity, making cells ready to lose polarity, to dissociate from their neighbors, and to migrate. Of note, downregulation of E-cadherin in renal tubular epithelium in obstructed kidney in vivo dominates at 7 days after UUO (2
), a timepoint that is significantly preceded by induction of ILK (Figure ). This implies that ILK could also be responsible for the disappearance of E-cadherin in renal tubules in fibrotic kidneys in vivo. The mechanism underlying E-cadherin suppression by ILK remains elusive. It has been previously shown that Snail transcription factor plays a critical role in suppressing E-cadherin expression in tumor epithelial cells (24
), and that ILK signaling is implicated in Snail expression (47
). However, no detectable Snail protein was observed in HKC cells under basal conditions after stimulation with TGF-β1 or following overexpression of ILK (Figure ), suggesting that Snail expression is cell content–dependent. Hence, it is clear that ILK mediates TGF-β-1–triggered E-cadherin repression by a mechanism independent of Snail in tubular epithelial cells.
Another hallmark for tubular EMT is cells beginning to overproduce ECM components and to properly assemble in the extracellular compartment, leading to excess accumulation of ECM, causing massive tissue fibrosis as seen in diseased kidney. The present study has underscored that ILK is essential in both fibronectin gene expression and its deposition into ECM (Figure ). The fact that both mRNA and cellular protein levels of fibronectin are increased in ILK-overexpressing cells indicates that ILK signaling is capable of influencing fibronectin gene expression. Nevertheless, judging from the magnitude of the induction of fibronectin extracellular assembly versus fibronectin expression (Figure ), it is easy to recognize that ILK preferentially promotes fibronectin assembly. ILK localizes to both focal adhesions and fibrillar adhesions (so-called ECM contacts) (16
), consistent with a role for ILK in fibronectin matrix assembly. Because these contacts are active sites for the deposition of fibronectin into ECM (48
), this pattern of ILK localization underscores a direct role in promoting fibronectin assembly. Given a role for ILK in connecting integrins and actin cytoskeleton, it is likely that ILK promotes the deposition of fibronectin into ECM by influencing the activation of integrins and/or by providing a molecular scaffold for the assembly of integrins that mediate fibronectin assembly and the actin cytoskeleton–associated proteins (39
). Regardless of the mechanism involved, our results establish that ILK is one crucial element in the cellular control of the deposition of fibronectin into ECM.
To complete tubular EMT in vivo, the transformed cells have to migrate into the interstitial compartment of the kidney. This task apparently requires coordinated actions to disrupt the TBM that normally confines tubular epithelial cells, and to allow the transformed cells to finally enter the interstitium. In this regard, it is of interest to point out that ILK is also involved in the regulation of cell migration, cell motility, and invasion in three-dimensional matrix. The observation that forced expression of ILK induces MMP-2 expression in tubular epithelial cells not only highlights that ILK can mimic TGF-β1’s action (2
), but also suggests that MMP-2 may be responsible for TBM degradation and for the invasive phenotype of the transformed cells. Consistently, recent studies also indicate that ILK induces an invasive phenotype in brain tumor cell lines via AP-1–dependent upregulation of MMP-9 expression (52
). In addition, its ability to phosphorylate myosin light chain may also hint at an important role for ILK in regulating cell motility (53
). Accordingly, ILK induction in renal tubules is specifically localized at the basal region close to TBM in the early stage of obstructive injury (Figure ), suggesting a crucial role of ILK in TBM degradation in the fibrotic kidney in vivo.
In view of an important role of ILK in mediating tubular EMT as described above, it is not difficult to envision a central role for ILK in the pathogenesis of renal fibrosis. Consistent with this view, emerging evidence suggests that dysregulation of ILK is implicated in various chronic glomerular diseases. For instance, ILK induction is found in glomerular podocytes, which is associated with progressive podocyte failure leading to proteinuria in animal models and in patients (21
). It is also shown that increased ILK expression in mesangial cells is associated with diabetic glomerulosclerosis (22
). The results in this study expand the correlation between ILK dysregulation and chronic renal fibrosis beyond the glomeruli. We have shown a tubule-specific induction of ILK in mouse kidney after both obstructive and diabetic injury, and such tubular ILK induction is closely associated with EMT and interstitial fibrosis in these models (2
). Of note, a dramatic induction of ILK (about 18-fold) in obstructive nephropathy (Figure ) is consistent with a high incidence of EMT in this aggressive form of renal fibrosis (2
). In diabetic nephropathy, a moderate induction of ILK (about threefold) is associated with the low prevalence of EMT in the early phase of tubulointerstitial lesions in this model (54
). Therefore, the frequency of tubular EMT in diseased kidneys is proportional to, and perhaps dictated by, the magnitude of ILK induction in renal epithelial cells after injury. In agreement with this notion, inhibition of ILK expression by HGF dramatically blocks tubular EMT and ameliorates renal fibrosis in obstructive nephropathy (Figure ) (6
). Hence, we propose a causative relationship between ILK upregulation and the activation of matrix-producing fibroblasts via EMT in renal fibrosis. More importantly, our study suggests a linear pathway that couples TGF-β1, Smad signaling, ILK, tubular EMT, and renal interstitial fibrogenesis.
Given the facts that a dominant-negative, kinase-dead form of ILK largely abolishes TGF-β1–induced tubular EMT and that inhibition of ILK expression by HGF blocks tubular EMT and reduces renal fibrosis, ILK signaling could be exploited as a novel therapeutic target for designing new treatment regimens for patients with chronic renal insufficiency. It is tempting to speculate that new strategies aimed at ILK expression and signaling may be effective at intercepting the profibrotic actions of TGF-β1 and thereby halting the onset and progression of chronic renal fibrosis. Therefore, further studies are warranted to develop molecular tools that downregulate ILK expression (such as HGF) or disrupt ILK function (55
), which allows us to intervene in ILK signaling for blockade of tubular EMT and for ultimate amelioration of the progressive loss of renal function in the fibrotic kidney.