Macrophage Matrix Metalloproteinase-9 Mediates Epithelial-Mesenchymal Transition in Vitro in Murine Renal Tubular Cells

As a rich source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages are well-placed to play an important role in renal fibrosis. However, the exact underlying mechanisms and the extent of macrophage involvement are unclear. Tubular cell epithelial−mesenchymal transition (EMT) is an important contributor to renal fibrosis and MMPs to induction of tubular cell EMT. The aim of this study was to investigate the contribution of macrophages and MMPs to induction of tubular cell EMT. The murine C1.1 tubular epithelial cell line and primary tubular epithelial cells were cultured in activated macrophage-conditioned medium (AMCM) derived from lipopolysaccharide-activated J774 macrophages. MMP-9, but not MMP-2 activity was detected in AMCM. AMCM-induced tubular cell EMT in C1.1 cells was inhibited by broad-spectrum MMP inhibitor (GM6001), MMP-2/9 inhibitor, and in AMCM after MMP-9 removal by monoclonal Ab against MMP-9. AMCM-induced EMT in primary tubular epithelial cells was inhibited by MMP-2/9 inhibitor. MMP-9 induced tubular cell EMT in both C1.1 cells and primary tubular epithelial cells. Furthermore, MMP-9 induced tubular cell EMT in C1.1 cells to an extent similar to transforming growth factor-β. Transforming growth factor-β-induced tubular cell EMT in C1.1 cells was inhibited by MMP-2/9 inhibitor. Our in vitro study provides evidence that MMPs, specifically MMP-9, secreted by effector macrophages can induce tubular cell EMT and thereby contribute to renal fibrosis.Interstitial macrophage infiltration is a hallmark of all progressive renal diseases regardless of the initial cause of the injury.^1,2 Macrophages have long been known to play an important role in renal fibrosis,³ which is a central component of the final common pathway leading to renal failure. Previous studies have demonstrated a close association between macrophage infiltrate and excessive extracellular matrix protein accumulation in diseased human kidney as well as in experimental models.^4–6 In addition, the number of infiltrating macrophages has been shown to correlate well with the number of myofibroblasts,^7,8 the effector cells responsible for secretion of extracellular matrix proteins. A recent study revealed that blockade of macrophage recruitment in obstructive renal injury resulted in a reduction in renal fibrosis via tubular cell epithelial−mesenchymal transition (EMT),⁹ which has been recognized as an important source of myofibroblasts in renal fibrosis. However, the exact mechanism underlying the contribution of macrophages to renal fibrosis via tubular cell EMT remains undefined. As a major source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages may be major determinants of the outcome of renal fibrosis.Tubular cell EMT is a process by which tubular epithelial cells lose their epithelial characteristics and acquire a mesenchymal phenotype. This process has been recognized as one of several pathways contributing to the myofibroblast population in renal fibrosis.¹⁰ Despite emerging and conflicting evidence about the relative importance of various sources of myofibroblasts,^11,12 it is generally accepted that tubular cell EMT plays an important role in renal fibrosis. Since the concept of tubular cell EMT was first proposed, numerous studies have provided evidence for tubular cell EMT in various experimental models as well as in human biopsies.¹⁰ Furthermore, the importance of tubular cell EMT has been demonstrated by Iwano et al¹³ using transgenic mice and direct genetic tagging of tubular epithelial cells to show that more than a third of myofibroblasts in kidneys with unilateral ureteral obstruction are derived from tubular epithelial cells via tubular cell EMT. Moreover, blockade of tubular EMT has been shown to attenuate renal fibrosis in obstructive nephropathy.¹⁴ However, some controversy remains as to whether tubular cell EMT plays a consistent role in other experimental models, and its exact contribution in renal fibrosis is yet to be established.Although pro-fibrogenic growth factors are well known as inducers of tubular cell EMT, cumulative evidence suggests an important role for MMPs. Traditionally, MMPs were thought to be antifibrogenic due to their ability to degrade extracellular matrix proteins, yet MMPs—in particular MMP-2 and MMP-9—have been recognized as promoters of tubular cell EMT via basement membrane disruption. In fact, induction of tubular cell EMT in vitro¹⁵ and in vivo¹⁴ has been shown to be associated with increased expression of MMP-2 and MMP-9. Earlier studies have demonstrated that tubular epithelial cells undergoing mesenchymal transition are closely associated with damaged tubular basement membrane and that complete transition requires tubular basement membrane damage.¹⁶ Later studies have shown directly that MMPs can disrupt basement membrane integrity; loss of MMP-9 expression lead to preservation of basement membrane integrity and inhibition of tubular cell EMT in obstructed kidney of tissue type plasminogen activator knockout mice.¹⁴ Despite this evidence supporting induction of tubular cell EMT by MMPs, the precise contribution of MMPs may have been underestimated. In cancer research, MMPs are well known to directly induce EMT in tumor cells of epithelial origin and to promote tumor progression via basement membrane disruption.¹⁷ MMP-2 has been shown consistently to be necessary and sufficient to induce tubular cell EMT in a rat tubular epithelial cell line (NRK52e).¹⁸ In addition, recent studies from our laboratory have demonstrated that MMP-3 and MMP-9 are also capable of inducing tubular cell EMT in NRK52e cells via the disruption of the cell adhesion molecule E-cadherin. Finally, the fact that transforming growth factor (TGF)-β-induced tubular cell EMT in NRK52e was inhibited by a broad spectrum MMP inhibitor suggests a primary role of MMP in TGF-β-induced tubular cell EMT.¹⁹ Together, these data suggest that MMPs from macrophages may play a major role in induction of tubular cell EMT. Therefore the aim of this study was to investigate the contribution of macrophages and their secreted MMPs to the induction of tubular cell EMT.