Plasmin(ogen) Promotes Renal Interstitial Fibrosis by Promoting Epithelial-to-Mesenchymal Transition

Abstract

Plasminogen (Plg) activator inhibitor-1 (PAI-1) is an important fibrosis-promoting molecule. Whether this effect can be attributed to PAI-1’s activity as an inhibitor of plasmin generation is debated. This study was designed to investigate the role of Plg in renal fibrosis using in vivo and in vitro approaches. Plg-deficient (Plg−/−) and wild-type (Plg+/+) C57BL/6 mice were subjected to unilateral ureteral obstruction or sham surgery (n = 8/group; sham, days 3, 7, 14, and 21). Plg deficiency was confirmed by the absence of Plg mRNA, protein, and plasmin activity. After 21 d of unilateral ureteral obstruction, total kidney collagen was significantly reduced by 35% in the Plg−/− mice. Epithelial-to-mesenchymal transition (EMT), as typified by tubular loss of E-cadherin and acquisition of α-smooth muscle actin, was also significantly reduced in Plg−/− mice, 76% and 50%, respectively. Attenuation of EMT and fibrosis severity in the Plg−/− mice was associated with significantly lower levels of phosphorylated extracellular signal–regulated kinase (ERK) and active TGF-β. In vitro, addition of plasmin (20 μg/ml) to cultures of murine tubular epithelial cells initiated ERK phosphorylation within minutes, followed by phenotypic transition to fibroblast-specific protein-1+, α-smooth muscle actin+, fibronectin-producing fibroblast-like cells. Both plasmin-induced ERK activation and EMT were significantly blocked in vitro by the protease-activated receptor-1 (PAR-1) silencing RNA; by pepducin, a specific anti–PAR-1 signaling peptide; and by the ERK kinase inhibitor UO126. Plasmin-induced ERK phosphorylation was enhanced in PAR-1–overexpressing tubular cells. These findings support important profibrotic roles for plasmin that include PAR-1–dependent ERK signaling and EMT induction.