resolvin-d1 and Ureteral-Obstruction

Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation.

Topics: Animals; Blotting, Western; Cell Proliferation; Cells, Cultured; Collagen; Culture Media, Conditioned; Docosahexaenoic Acids; Endothelium, Vascular; Fibroblasts; Fibrosis; Immunoprecipitation; Kidney Diseases; Luciferases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Nitric Oxide Synthase Type III; Phosphorylation; Promoter Regions, Genetic; Rats; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta1; Ureteral Obstruction

Resolvin E1 (RvE1) is a naturally occurring lipid-derived mediator generated during the resolution of inflammation. The anti-inflammatory effects of RvE1 have been demonstrated in a variety of disease settings; however, it is not known whether RvE1 may also exert direct anti-fibrotic effects. We examined the potential anti-fibrotic actions of RvE1 in the mouse obstructed kidney-a model in which tissue fibrosis is driven by unilateral ureteric obstruction (UUO), an irreversible, non-immune insult. Administration of RvE1 (300 ng/day) to mice significantly reduced accumulation of α-smooth muscle actin (SMA)(+) myofibroblasts and the deposition of collagen IV on day 6 after UUO. This protective effect was associated with a marked reduction of myofibroblast proliferation on days 2, 4 and 6 after UUO. RvE1 treatment also inhibited production of the major fibroblast mitogen, platelet-derived growth factor-BB (PDGF-BB), in the obstructed kidney. Acute resolvin treatment over days 2-4 after UUO also had a profound inhibitory effect upon myofibroblast proliferation without affecting the PDGF expression, suggesting a direct effect upon fibroblast proliferation. In vitro studies established that RvE1 can directly inhibit PDGF-BB-induced proliferation in primary mouse fibroblasts. RvE1 induced transient, but not sustained, activation of the pro-proliferative ERK and AKT signalling pathways. Of note, RvE1 inhibited the sustained activation of ERK and AKT pathways seen in response to PDGF stimulation, thereby preventing up-regulation of molecules required for progression through the cell cycle (c-Myc, cyclin D) and down-regulation of inhibitors of cell cycle progression (p21, cip1). Finally, siRNA-based knock-down studies showed that the RvE1 receptor, ChemR23, is required for the anti-proliferative actions of RvE1 in cultured fibroblasts. In conclusion, this study demonstrates that RvE1 can inhibit fibroblast proliferation in vivo and in vitro, identifying RvE1 as a novel anti-fibrotic therapy.

Topics: Animals; Becaplermin; Cell Proliferation; Docosahexaenoic Acids; Eicosapentaenoic Acid; Fibroblasts; Fibrosis; Kidney; Macrophages; Male; Mice, Inbred C57BL; Primary Cell Culture; Proto-Oncogene Proteins c-sis; Ureteral Obstruction