Content area
Abstract
Renal fibrosis leads to chronic kidney disease (CKD), which affects over 15% of the world population. There is a critical need for the development of effective therapeutic regimens for CKD. Two major contributing factors to CKD progression are epithelial dysfunction and fibrotic paracrine signaling. Epithelial dysfunction is characterized by dedifferentiation, proliferative arrest, apoptosis and fibrotic factor/matrix deposition. Secretion of pro-fibrotic/inflammatory paracrine factors by fibrotic G2/M growth arrested tubular cells can trigger maladaptive responses in neighboring epithelial and fibroblast cells. Plasminogen activator inhibitor-1 (PAI-1) is a causative factor for fibrosis, as global ablation in mice affords protection from several nephropathies. A surprising recognition that PAI-1 contribution to fibrosis likely occurs via uPA-independent mechanisms prompted us to reexamine its pathogenic role. Since tubular PAI-1 expression is highly upregulated in various kidney diseases, we hypothesize that PAI-1 promotes tubular dysfunction. Human renal epithelial (HK2) cells stably overexpressing PAI-1 spontaneously undergo dedifferentiation (loss of E-cadherin and gain of vimentin), G2/M growth arrest (p21 and pHistone3 upregulation), with robust induction of fibronectin, collagen-1 and CTGF/CCN2. PAI-1-transduced cells are also susceptible to apoptosis (cleaved-caspase3 and annexin-V positivity) compared to vector-controls, demonstrating a previously unknown role for PAI-1 in fibrotic tubular maladaptive repair. Mechanistically, persistent PAI-1 expression results in a loss of klotho expression, p53 upregulation, increased TGF-β1-Receptor-I/II levels and SMAD3 phosphorylation. Vector-driven restoration of klotho in PAI-1-transductants, in fact, attenuated fibrogenesis and reversed the proliferative defects, identifying a novel role for PAI-1 in klotho loss in renal disease. Additionally, genetic suppression of p53 reversed the PAI-1-driven maladaptive repair, confirming a pathogenic role of p53 upregulation in this context and uncovering a novel role for PAI-1 in promoting renal p53 signaling. Genetic or chemical inhibition of TGF-β-RI also attenuated the PAI-1-driven fibrotic phenotype, independent of TGF-β1 ligand synthesis. Using unbiased cytokine array profiling, we identified pro-inflammatory factor osteopontin (OPN) as a key regulator of PAI-1-dependent epithelial dysfunction. OPN was significantly increased in PAI-1 transductants via klotho and TGF-βRI-dependent mechanisms. OPN gene depletion repressed PAI-1-driven fibrotic responses, proliferative defects and upregulation of p53 and Twist transcription factors. Twist silencing in PAI-1 overexpressing transductants also attenuated subsequent renal epithelial cell pathologies. Collectively, this work identifies a novel role for PAI-1 in promoting fibrotic tubular dysfunction, and these autonomous effects are orchestrated via a klotho-TGF-βRI-OPN-p53-Twist signaling axis. This study also identifies a novel role for PAI-1 in pathogenic renal cell-cell communication. Dysfunctional PAI-1 overexpressing tubular cells synthesize and secrete various paracrine factors (including OPN, IL-6 and IL-8), which promoted dedifferentiation and fibrotic factor expression in non-injured renal epithelial cells (epithelial-epithelial crosstalk) and triggers a fibro-proliferative response in primary human kidney fibroblasts (epithelial-fibroblast crosstalk). These PAI-1-induced non-autonomous effects mediated by soluble factors likely exacerbate tubular injury and promote excessive extracellular matrix remodeling. Therefore, targeting renal PAI-1 upregulation could serve as a novel CKD therapeutic strategy to inhibit both autocrine (tubular dysfunction) and non-autonomous paracrine signaling cascades (cell-cell communication) that perpetuate fibrosis progression.
