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.
Highlights
Renal interstitial fibrosis is the common final pathway in the progression of end stage kidney disease, irrespective of the initial trigger or site of injury
Expression of c-myc became evident at 12 hr after ureteric obstruction (UUO) while there was a modest reduction in p21 levels at 6 hr and a substantial reduction in p21 levels at 48 hr after UUO (Fig 1C&D), consistent with previous studies showing that proliferation of interstitial myofibroblasts and tubular cells becomes evident at 48 hrs after UUO [34]
Immunoprecipitation identified a direct interaction between active Jun amino terminal kinase (JNK) and Smad3 at 6 hr after UUO (Fig 2A&B), suggesting a role for JNK in phosphorylating the Smad3 linker region in the development of renal fibrosis
Summary
Renal interstitial fibrosis is the common final pathway in the progression of end stage kidney disease, irrespective of the initial trigger or site of injury. The contribution of tubular epithelial cells, fibroblast/myofibroblasts and inflammation to the development of interstitial fibrosis has been extensively studied. Recent studies have shown that injury to glomerular endothelial cells directly contributes to podocyte and mesangial cell damage and the development of glomerulosclerosis [2,3,4,5]. We have shown that injury to glomerular endothelial cells precedes podocyte damage and the development of proteinuria and glomerulosclerosis in adriamycin-induced nephropathy, while nitric oxide synthase 3 (NOS3/eNOS) deficiency accelerates kidney injury in this model [6]. Interesting, conditioned media from NOS3 overexpressing endothelial cells protects cultured podocytes from TNFa-induced injury [6], prompting us to investigate the potential role of endothelial cell injury on fibroblasts/myofibroblasts in the development of renal interstitial fibrosis
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