Endothelial dysfunction exacerbates renal interstitial fibrosis through enhancing fibroblast Smad3 linker phosphorylation in the mouse obstructed kidney

PLoS One. 2013 Dec 31;8(12):e84063. doi: 10.1371/journal.pone.0084063. eCollection 2013.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Proliferation
  • Cells, Cultured
  • Collagen / genetics
  • Collagen / metabolism
  • Culture Media, Conditioned / pharmacology
  • Docosahexaenoic Acids / pharmacology
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / metabolism
  • Endothelium, Vascular / pathology*
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Fibroblasts / pathology*
  • Fibrosis / drug therapy
  • Fibrosis / metabolism
  • Fibrosis / pathology*
  • Immunoprecipitation
  • Kidney Diseases / drug therapy
  • Kidney Diseases / metabolism
  • Kidney Diseases / pathology*
  • Luciferases / metabolism
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Mutation / genetics
  • Nitric Oxide Synthase Type III / physiology*
  • Phosphorylation
  • Promoter Regions, Genetic
  • Rats
  • Signal Transduction
  • Smad3 Protein / genetics
  • Smad3 Protein / metabolism*
  • Transforming Growth Factor beta1 / metabolism
  • Ureteral Obstruction / drug therapy
  • Ureteral Obstruction / metabolism
  • Ureteral Obstruction / pathology*

Substances

  • Culture Media, Conditioned
  • Smad3 Protein
  • Smad3 protein, mouse
  • Transforming Growth Factor beta1
  • resolvin D1
  • Docosahexaenoic Acids
  • Collagen
  • Luciferases
  • Nitric Oxide Synthase Type III
  • Nos3 protein, mouse

Grants and funding

This study was supported by Monash University Accelerating Programme and the National Health and Medical Research Council (NHMRC) of Australia. JL is the recipient of a NHMRC Career Development Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.