Chronic kidney disease (CKD) and failure are problems of increasing importance. Regardless of the primary etiology, CKD is characterized by tubular atrophy, interstitial fibrosis, and glomerulosclerosis. It has been assumed that diminished matrix metalloproteinase (MMP) activity is responsible for the accumulation of the extracellular matrix (ECM) proteins and collagens that typify the fibrotic kidney. Here we demonstrate that transgenic renal proximal tubular epithelial expression of a specific enzyme, MMP-2, is sufficient to generate the entire spectrum of pathological and functional changes characteristic of human CKD. At the earliest point, MMP-2 leads to structural alterations in the tubular basement membrane, a process that triggers tubular epithelial-mesenchymal transition, with resultant tubular atrophy, fibrosis and renal failure. Inhibition of MMP-2, specifically in the early, prefibrotic stages of disease may offer an additional approach for treatment of these disabling disorders.

The matrix metalloproteinases (MMPs), 1 which include the interstitial collagenases, stromelysins 1-3, matrilysin, 92-kDa gelatinase B (MMP-9), and the 72-kDa gelatinase A (MMP-2), constitute a family of matrix-degrading enzymes with distinctive extracellular matrix substrate specificities. These enzymes are characterized by activity at neutral pH, secretion in latent, proenzyme forms, dependence upon zinc for catalytic activity, and inhibition by tissue inhibitors of metalloproteinases 1-3 (1). While MMP-2 and MMP-9 share similar substrate specificities, encompassing type IV and V collagen, fibronectin, and laminin (2, 3), their patterns of expression are distinct. MMP-2 has been the focus of considerable interest, since high level expression of this enzyme in metastatic tumor cells correlates with overall metastatic potential (4, 5), presumably due to enhanced degradation of the major basal membrane component, Type IV collagen.Furthermore, MMP-2 is of importance in inflammatory reactions found in numerous tissues, including the kidney. The development of the sclerotic lesion in chronic glomerulosclerosis is temporally correlated with high level expression of MMP-2 (6). Remarkably, MMP-2 acts not only as a matrixdegrading enzyme but also directly stimulates growth by glomerular mesangial cells (MC) (7) with assumption of an inflammatory phenotype characterized by increased synthesis of prosclerotic interstitial collagens.Although in most cell culture systems MMP-2 expression is constitutive in nature, there are cells described with a differentially regulated expression of MMP-2 both in vivo and in vitro. For example, MMP-2 is highly expressed in a temporally limited pattern in the developing murine lung and kidney (8).In vitro, MMP-2 expression by MCs can be stimulated with interleukin-1␤, tumor necrosis factor-␣, and transforming growth factor-␤ (9). The 5Ј-flanking region up to Ϫ1686 bp relative to the translation start site of the rat MMP-2 gene was recently evaluated for the existence of MC-specific regulatory elements (10), with the resultant identification of an apparently unique, 40-bp cis-acting enhancer element located at Ϫ1282 to Ϫ1322 bp relative to the translation start site, designated MMP-2 RE1. The MMP-2 RE1 confers cell-specific transactivation of luciferase reporter constructs containing MMP-2 RE1 in conjunction with either the homologous MMP-2 or a heterologous SV40 promoter. Furthermore, specific DNA binding activity to this element was demonstrated in MC nuclear extracts (10). Although nuclear protein binding activity to this enhancer element was also detected in other cell types, positive transactivation in conjunction with the homologous MMP-2 promoter was confined to MCs.

The development of progressive glomerulosclerosis in the renal ablation model has been ascribed to a number of humoral and hemodynamic events, including the peptide growth factor, transforming growth factor-j31 (TGF-fi1).An important role has also been attributed to angiotensin II (All), which, in addition to its hemodynamic effects, can stimulate transcription of TGF-fi1. We postulated that increased glomerular production of All, resulting from enhanced intrinsic angiotensinogen expression, stimulates local TGF-131 synthesis, activating glomerular matrix protein synthesis, and leads to sclerosis. Using in situ reverse transcription, the glomerular cell sites of a-l(IV) collagen, fibronectin, laminin B1, angiotensinogen, and TGF-fi1 mRNA synthesis were determined at sequential periods following renal ablation. The early hypertrophic phase was associated with global, but transient, increases in the mRNA for a-1 (IV) collagen. No changes were noted for fibronectin, TGF-j31, and angiotensinogen mRNAs. At 24 d after ablation, at which time sclerosis is not evident, endothelial cells, particularly in the dilated capillaries at the vascular pole, expressed angiotensinogen and TGF-131 mRNAs, as well as fibronectin and laminin B1 RNA transcripts. By 74 d after ablation angiotensinogen and TGF-,B1 mRNAs were widely distributed among endothelial and mesangial cells, and were particularly prominent in regions of evolving sclerosis. These same regions were also notable for enhanced expression of matrix protein mRNAs, particularly fibronectin. All receptor blockade inhibited angiotensinogen, TGF-,B1, fibronectin, and Ia ni n B1 mRNA expression by the endothelium. We conclude that, as a result of hemodynamic changes, injured or activated endothelium synthesizes angiotensinogen, triggering a cascade of TGF-,B1 and matrix protein gene expression with resultant development of the segmental glomerular sclerotic lesion. (J. Clin. Invest. 1995. 96:953-964.)

A biologic role for the 72-kDa gelatinase A (matrix metalloproteinase 2; MMP-2), beyond simple extracellular matrix turnover, was evaluated in glomerular mesangial cells. To determine the significance of MMP-2 secretion for the acquisition of the inflammatory phenotype, we reduced the constitutive secretion of MMP-2 by cultured mesangial cells with antisense RNA expressed by an episomally replicating vector or with specific anti-MMP-2 ribozymes expressed by a retroviral transducing vector. The phenotype of the transfected, or retrovirally infected, cells was profoundly altered from the activated state and closely approximated that of quiescent cells in vivo. The prominent differences included a change in the synthesis and organization of the extracellular matrix, loss of activation markers, and a virtually total exit from the cell cycle. Reconstitution with exogenous active, but not latent MMP-2, induced a rapid return to the inflammatory phenotype in vitro. This effect was specific to MMP-2, because the closely related MMP-9 did not reproduce these changes. Furthermore, this pro-inflammatory effect of MMP-2 is dependent upon the active form of the enzyme, which can be produced by an autocatalytic activation process on the mesangial cell plasma membrane. It is concluded that MMP-2 acts directly upon mesangial cells to permit the development of an inflammatory phenotype. Specific inhibition of MMP-2 activity in vivo may represent an alternate means of ameliorating complex inflammatory processes by affecting the phenotype of the synthesizing cells, per se.