u-0126 and Ureteral-Obstruction

Interleukin (IL)-17A is upregulated in several renal diseases and plays a crucial role in renal inflammation. However, it remains unclear how IL-17A contributes to renal fibrosis. Our result demonstrated that IL-17A expression was upregulated in the obstructed kidney of unilateral ureter obstruction (UUO) mice when compared to the contralateral control kidney. Inhibition of IL-17A functions by the intravenous administration of an anti-IL-17A receptor antibody (100 μg) 2 h prior to UUO and on post-UUO day 1 and 3 significantly reduced fibronectin expression in the UUO kidney. The addition of IL-17A (25-100 μg) to human renal proximal tubular cells or renal fibroblasts caused an increase in fibronectin production and extracellular signal-regulated kinase (ERK)1/2 activation, which were reduced upon pretreatment with the ERK inhibitor U0126. The level of phosphorylated (p)-ERK1/2 was increased in the UUO kidney, but reduced by the administration of the anti-IL-17A receptor antibody, verifying the importance of the ERK pathway in vivo. TGF-β1 mRNA expression and protein were increased in the UUO kidney and in IL-17A-stimulated cultured cells. The administration of an anti-TGF-β1 neutralizing antibody or TGF-β1 receptor I inhibitor (SB431542) to cells abrogated the IL-17A-mediated increase of fibronectin production. IL-17A induced an increase in p-Smad2 and p-Smad3 expression at 7.5 min and 24 h and pretreatment with the anti-TGF-β1 neutralizing antibody, and SB431542 reduced the IL-17A-stimulated increase of p-Smad2. Knockdown of Smad2 or Smad3 expression inhibited the IL-17A-enhanced production of fibronectin. These results suggest an essential role for the TGF-β/Smad pathway in the IL-17A-mediated increase of fibronectin production. This study demonstrates that IL-17A contributes to the production of extracellular matrix, and targeting its associated signaling pathways could provide a therapeutic target for preventing renal fibrosis.

Topics: Animals; Benzamides; Butadienes; Cell Line; Cytokines; Dioxoles; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Fibronectins; Fibrosis; Humans; Interleukin-17; Kidney; Mice; Mice, Inbred BALB C; Models, Animal; Nitriles; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1; Ureteral Obstruction

A number of growth factors have been shown to induce proliferation of renal cell types in animal models of kidney disease. In vitro studies suggest that many such growth factors induce renal cell proliferation through the extracellular signal-regulated kinase (ERK) pathway. The aim of this study was to determine the functional role of ERK signalling in cell proliferation in the obstructed kidney.. Unilateral ureteric obstruction was induced in C57BL/6J mice which then received an ERK inhibitor drug (U0126 100 mg/kg t.i.d.), vehicle (DMSO) or no treatment, starting at day 2 after unilateral ureteric obstruction surgery and continuing until animals were killed on day 5. Cell proliferation was assessed by uptake of bromodeoxyuridine (BrdU).. In normal mice, phosphorylation (activation) of ERK (p-ERK) was restricted to collecting ducts. Western blotting identified a marked increase in p-ERK in the obstructed kidney in the no-treatment and vehicle-treated groups. Immunostaining showed strong p-ERK staining in many tubules and in interstitial cells. U0126 treatment inhibited ERK phosphorylation as assessed by western blot and immunostaining. The number of BrdU+ cortical tubular cells was reduced by vehicle treatment but was not further changed by U0126 treatment. In contrast, interstitial cell proliferation in the obstructed kidney was unaltered by vehicle treatment, but this was significantly inhibited by U0126. This was associated with a reduction in interstitial macrophage accumulation, but no effect was seen upon interstitial accumulation of alpha-SMA+ myofibroblasts. Renal fibrosis, as assessed by collagen deposition, was unaffected by U0126 or vehicle treatment.. These studies show that accumulation of interstitial macrophages in the obstructed kidney is, in part, dependent upon the ERK signalling pathway.

Topics: Animals; Butadienes; Cell Proliferation; Enzyme Inhibitors; Kidney Diseases; Macrophages; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase Kinases; Nitriles; Signal Transduction; Ureteral Obstruction

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 alpha-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-beta. In vitro, addition of plasmin (20 microg/ml) to cultures of murine tubular epithelial cells initiated ERK phosphorylation within minutes, followed by phenotypic transition to fibroblast-specific protein-1+, alpha-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.

Topics: Actins; Animals; Butadienes; Cadherins; Cell Movement; Collagen; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Fibrinolysin; Fibrosis; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitriles; Phosphorylation; Plasminogen Activator Inhibitor 1; Receptor, PAR-1; Signal Transduction; Transforming Growth Factor beta; Ureteral Obstruction