| Renal fibrosis, the common feature of most chronic kidney diseases (CKDs), is characterized by glomerulosclerosis and tubulointerstitial fibrosis. The progression of renal fibrosis may lead to the continuously loss of functional renal parenchyma that finally contribute to end-stage kidney failure. There are various major causes that can induce renal fibrosis, among which, the dysfunction of renal tubular epithelial cells has been identified contribute to renal fibrosis. Although there is a debate on type Ⅱ epithelial-to-mesenchymal transition (EMT), which indicates that injured epithelial cell can be the direct precursor of myofibroblast, tubular epithelial cells undergoing pro-fibrotic cellular changes including increased expression of transforming growth factor-β1 (TGF-β1) and extracellular matrix (ECM) deposition can contribute to the early development and progression of renal interstitial fibrosis under multiple pathological conditions.A number of proteins, cytokine and growth factors may be involved in the progression of renal interstitial fibrosis. Increasing evidence suggests that angiotensin Ⅱ (Ang Ⅱ), one of the primary intermediate of rennin-angiotensin system (RAS), is capable of promoting further inflammation and fibrosis under pathophysiological circumstances. Ang Ⅱ is one of the main pathogenetic mediators of renal interstitial fibrosis in various kidney diseases including obstructive nephropathy. A number of previous studies have revealed that suppression of intrarenal RAS prevents the pro-fibrotic changes in unilateral ureteral obstruction (UUO) model.The Janus kinase (JAK) family/signal transducers and activators of transcription (STAT) signaling pathway is thought to contribute to a pleiotropic cascade essential for a wide range of signal transduction cytokines and growth factors expression. JAK2/STAT3 is one of the best characterized signaling pathways confirmed to be the downstream mediator of Ang Ⅱ. STAT3 has been shown to play a crucial role in promoting cell cycle progression, and preventing apoptosis. Over the past several years, STAT3 has increasingly been implicated in the progression of renal fibrogenesis, especially in the obstructed kidney. Either Ang Ⅱ or STAT3 is confirmed to mediate kidney disfunction separately. However, the role of Ang Ⅱ-STAT3 signaling pathway in renal fibrogenesis is still uncertain.STAT3 activation requires phosphorylation on tyrosine 705 (Tyr705) and serine 727 (Ser727). It can also be reversibly acetylated on lysine 685 (Lys685). The acetylation of this single lysine residue regulated by histone acetyltransferase (HAT) cAMP response element binding protein-binding protein (CBP)/p300 and histone deacetylases (HDACs) contributes significantly to STAT3 dimerization and transcription activity.Transcriptional coactivator p300 and its homolog CBP are capable of regulating many cellular processes via its substrate and downstream coactivator, such as STAT3. Furthermore, there is strong evidence demonstrating that p300 promotes the accumulation of fibronectin, a hallmark protein of ECM which associates with cell adhesion and fibrosis.Several HDACs have been reported to be involved in deacetylation of STAT3. One of the best characterized deacetylases is silent mating type information regulation 2 homolog 1 (SIRT1), a member of nicotinamide adenine dinucleotide (NAD+)-dependent class Ⅲ protein deacetylases inhibited by nicotinamide. SIRT1, which mediates deacetylation of STAT3 Lys685 sit, can be activated by resveratrol, a natural polyphenol with renal protective effects that depend on deacetylation of Smad3, p53, and NF-κB p65. However, it remains to be clarified whether inhibition of STAT3 acetylation is involved in antifibrotic action of resveratrol.In the present study, we investigated the relationship between acetylation and phosphorylation of STAT3 in Ang Ⅱ-induced pro-fibrotic responses in renal tubular epithelial cells and UUO-induced renal fibrosis to determine the role of STAT3 signaling in renal fibrogenesis both in vitro and in vivo. Results:Part Ⅰ. The role of STAT3 activation in AngⅡ induced pro-fibrotic responses in renal tubular epithelial cells1. Ang Ⅱ induced pro-fibrotic responses renal tubular epithelial cellsWestern blot analysis showed that treatment of cells with Ang Ⅱ for 48 hours increased fibronectin, collagen Ⅳ and α-smooth muscle actin (α-SMA) protein levels in a dose-dependent manner in NRK-52E cells. Ang Ⅱ at 1 μM significantly up-regulated these pro-fibrotic hallmarks.2. Ang Ⅱ induced STAT3 activation in renal tubular epithelial cellsThe investigation on the possible intracellular molecular cascades demonstrated that JAK2/STAT3 signaling pathway was activated in NRK-52E tubular epithelial cells after Ang II application. JAK2 phosphorylation was up-regulated at 30 min after Ang II treatment. STAT3 phosphorylation on Tyr705 was also enhanced, peaking at 60 minutes after Ang II treatment. Ang II did not affect STAT3 phosphorylation on Ser727 or acetylation on Lys685 which was constitutively observed in NRK-52E cells.3. Curcumin inhibited STAT3 acetylation and phosphorylation in tubular epithelial cellsTo examine whether the JAK2/STAT3 signaling pathway is involved in Ang II-induced pro-fibrotic responses, curcumin, an inhibitor of p300 and JAK2, was recruited in the experiment. Western blot analysis showed that pretreatment of cells with curcumin (12.5 μM) for 60 minutes reduced STAT3 acetylation level and prevented Ang Ⅱ-induced up-regulation of STAT3 phosphorylation on Tyr705.4. Curcumin opposed pro-fibrotic responses induced by Ang Ⅱ in tubular epithelial cellsWestern blot analysis showed that curcumin (12.5 μM) also attenuated Ang II-driven up-regulation of fibronectin, collagen IV and TGF-β1.These results indicated that STAT3 pathway was involved in Ang II-induced pro-fibrotic responses in tubular epithelial cells.Part Ⅱ. The role of STAT3 acetylation on Lys685 and phosphorylation on Tyr705 in Ang Ⅱ-induced pro-fibrotic responses in renal tubular epithelial cells1. The inhibition of STAT3 acetylation on Lys685 opposed STAT3 phosphorylation on Tyr705 regulated by Ang Ⅱ in tubular epithelial cellsWestern blot analysis showed that either resveratrol (12.5 μM) or p300 (50 nM) siRNA decreased STAT3 acetylation on Lys685 and abolished Ang II-stimulated up-regulation of STAT3 phosphorylation on Tyr705, but not the phosphorylation on Ser727. Fluorescence immunocytochemistry results showed that pretreatment of cells with C646 (10 μM), a selective inhibitor of p300, rapidly inhibited Ang II-induced STAT3 nuclear translocation, which was proportional to phosphorylation on Tyr705.2. The inhibition of STAT3 acetylation on Lys685 opposed pro-fibrotic responses regulated by Ang Ⅱ in tubular epithelial cellsWestern blot analysis showed that either resveratrol (12.5 μM) or C646 (10 μM) attenuated Ang Ⅱ-induced up-regulation of fibronectin, collagen Ⅳ, TGF-β1 and α-SMA in renal tubular epithelial cells.3. The inhibition of STAT3 phosphorylation on Tyr705 had no effect on STAT3 acetylation on Lys685Western blot analysis showed that AG490 (12.5 μM), a tyrosine kinase tyrphostin which inhibits the activity of JAK2, suppressed Ang Ⅱ-induced STAT3 phosphorylation on Tyr705. However, AG490 did not affect STAT3 phosphorylation on Ser727 or STAT3 acetylation on Lys685.4. The inhibition of STAT3 phosphorylation on Tyr705 opposed STAT3-dependent pro-fibrotic responses induced by Ang Ⅱ in tubular epithelial cellsWestern blot analysis showed that pretreatment of cells with AG490 (12.5 μM) also abolished downstream fibronectin up-regulation induced by Ang Ⅱ in tubular epithelial cells.5. Up-regulation of STAT3 acetylation inforced STAT3 phosphorylation and downstream pro-fibrotic responses in renal tubular epithelial cells5.1 Nicotinamide induced STAT3 activation and pro-fibrotic responses in renal tubular epithelial cellsWestern blot analysis showed that nicotinamide, an inhibitor of SIRTs, significantly enhanced STAT3 acetylation on Lys685 and phosphorylation on Tyr705 in NRK-52E cells (48 h). These effects were accompanied by accumulation of fibronectin and collagen Ⅳ.5.2 p300 induced STAT3 activation and pro-fibrotic response in tubular epithelial cellsWestern blot analysis showed that overexpression of p300 by the transfection of a recombinant plasmid carrying p300 gene increased p300 protein level significantly in NRK-52E cells, accompanied by up-regulation of STAT3 acetylation on Lys685 and phosphorylation on Tyr705. Furthermore, p300 also increased fibronectin, collagen Ⅳ and TGF-β1 protein levels significantly.These results indicated that inhibition of STAT3 acetylation is associated with suppression of Ang Ⅱ-induced STAT3 phosphorylation on Tyr705 and STAT3 mediated pro-fibrotic responses in renal tubular epithelial cells. STAT3 phosphorylation on Tyr705 was necessary for Ang Ⅱ-induced pro-fibrotic responses in renal tubular epithelial cells. Up-regulation of STAT3 acetylation inforced STAT3 phosphorylation and downstream pro-fibrotic responses in renal tubular epithelial cells.Part Ⅲ. Resveratrol inhibited STAT3 acetylation, phosphorylation and renal fibrosis in the obstructed kidney1. UUO induced STAT3 activation and pro-fibrotic responses in the kidneyWestern blot analysis showed that in the murine UUO model, a significant increase in STAT3 phosphorylation on Tyr705 was observed one day after surgery while no obvious change in STAT3 acetylation on Lys685 had been found. The fibrotic associated proteins including fibronectin, collagen Ⅳ and α-SMA were increased in UUO mice and showed statistical significance since the third day after surgery, which maintained to the end of the experiment (7 d).2. Resveratrol inbibited UUO-induced STAT3 activation and expression of pro-fibrotic genes in the obstructed kidneyWestern blot analysis showed that resveratrol treatment of UUO mice by gavage administration decreased STAT3 acetylation on Lys685. This effect was accompanied by a reduction in UUO-induced Tyr705 phosphorylation (1 d). Resveratrol also inhibited UUO-induced up-regulation of fibronectin, collagen IV and a-SMA protein levels in the obstructed kidney (3 d).3. Resveratrol attenuated tubulointerstitial fibrosis in UUO kidneyMasson’s trichrome staining and hematoxylin-eosin (HE) staining showed the histology changes of resveratrol treated obstructed kidneys. In vehicle and sham kidneys, no fibrosis staining was seen. The obstructed kidneys showed tubulointerstitial fibrosis at days 3 which was apparently ameliorated by resveratrol treatment.These results in line with in vitro findings suggested that STAT3 acetylation was involved in the pro-fibrotic action in the obstructed kidney.Summary:In vitro study showed that Ang Ⅱ induced an increase in STAT3 phosphorylation on Tyr705 through JAK2 activation, accompanied by enhanced expression of fibronectin, collagen Ⅳ and α-SMA in tubular epithelial cells; The inhibition of STAT3 acetylation on Lys685 opposed Ang Ⅱ-induced STAT3 phosphorylation on Tyr705, accompanied by decresed fibronectin, collagen Ⅳ, TGF-β1 and α-SMA levels regulated by Ang Ⅱ; The inhibition of STAT3 phosphorylation on Tyr705 opposed STAT3-dependent expression of fibronectin regulated by Ang Ⅱ, but did not affect STAT3 acetylation on Lys685; Up-regulation of STAT3 acetylation on Lys685 inforced STAT3 phosphorylation on Tyr705, accompanied by up-regulation of fibronectin, collagen Ⅳ and TGF-β1 expression. Furthermore, resveratrol attenuated STAT3 phosphorylation on Tyr705 and renal fibrosis in UUO mice through inhibition of STAT3 acetylation on Lys685. These results suggest that STAT3 acetylation is necessary for STAT3 signaling mediated renal fibrosis. |
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