GQ5 Hinders Renal Fibrosis in Obstructive Nephropathy by Selectively Inhibiting TGF-β-Induced Smad3 Phosphorylation

Abstract

TGF-β1, via Smad-dependent or Smad-independent signaling, has a central role in the pathogenesis of renal fibrosis. This pathway has been recognized as a potential target for antifibrotic therapy. Here, we identified GQ5, a small molecular phenolic compound isolated from the dried resin of Toxicodendron vernicifluum, as a potent and selective inhibitor of TGF-β1-induced Smad3 phosphorylation. In TGF-β1-stimulated renal tubular epithelial cells and interstitial fibroblast cells, GQ5 inhibited the interaction of Smad3 with TGF-β type I receptor (TβRI) by blocking binding of Smad3 to SARA, suppressed subsequent phosphorylation of Smad3, reduced nuclear translocation of Smad2, Smad3, and Smad4, and downregulated the transcription of major fibrotic genes such as α-smooth muscle actin (α-SMA), collagen I, and fibronectin. Notably, intraperitoneal administration of GQ5 in rats immediately after unilateral ureteral obstruction (UUO) selectively inhibited Smad3 phosphorylation in UUO kidneys, suppressed renal expression of α-SMA, collagen I, and fibronectin, and resulted in impressive renal protection after obstructive injury. Late administration of GQ5 also effectively attenuated fibrotic lesions in obstructive nephropathy. In conclusion, our results suggest that GQ5 hinders renal fibrosis in rats by selective inhibition of TGF-β1-induced Smad3 phosphorylation.

Keywords: GQ5; Smad3 phosphorylation; TGF-β1; fibrosis; obstructive nephropathy; renal.

Figure 1.

The structure of GQ5.

Figure 2.

GQ5 attenuates renal interstitial fibrosis in UUO. Rats receive daily intraperitoneal injection of vehicle or GQ5 (40 mg/kg per day) right after (d1) or 7 days (d7) after UUO, and are euthanized at 14 days. (A) Representative micrographs of hematoxylin and eosin (HE) and Masson’s trichrome staining demonstrate kidney injury in indicated groups. (B) Quantification of the number of infiltrated cells in renal interstitium. (C) Quantification of renal tubular interstitial fibrotic score. (D) Real-time PCR analyses for mRNA expression of α-SMA, collagen I, and fibronectin in the obstructed kidney. (E) Representative micrographs of the protein expression of α-SMA, collagen I, and fibronectin in the obstructed kidneys. (F) Representative bands (two cases) of Western blot analyses for the expression of α-SMA, collagen I, and fibronectin in the obstructed kidneys. *P<0.05 versus sham; ^#P<0.05 versus vehicle (n=6 for each group).

Figure 3.

GQ5 selectively inhibits TGF-β1–induced Smad3 phosphorylation. NRK52E and NRK49F cells are preincubated with GQ5 for 1 hour before TGF-β1 (10 ng/ml) treatment. Cells are harvested 1 hour after TGF-β1 stimulation, and cell lysates are immunoblotted. (A) GQ5 selectively inhibits TGF-β1–induced Smad3 phosphorylation in NRK52E cells. (B) GQ5 selectively inhibits TGF-β1–induced Smad3 phosphorylation in NRK49F cells. (C) GQ5 does not affect Smad4 or Smad7 protein expression. (D) GQ5 does not affect TGF-β1–induced phosphorylation of p38, PI3K or ERK. Data are expressed as the mean±SD of three independent experiments. P<0.05 in p-Smad3 expression in GQ5-treated cells (A and B; ANOVA). *P<0.05 versus untreated cells. Ctrl, control.

Figure 4.

GQ-5 selectively inhibits Smad3 phosphorylation in UUO. Rats receive daily intraperitoneal injection of vehicle or GQ5 (40 mg/kg per day) right after (d1) or 7 days (d7) after UUO and are euthanized 14 days after UUO. (A and B) Immunohistochemical staining (A) and Western blots (B) are performed to analyze the expression of p-Smad3 and p-Smad2 in UUO rats. (C) Western blots show that GQ5 does not affect the expression of Smad4 or Smad7. (D) Western blots show that GQ5 does not affect the phosphorylation of p38, PI3K, or ERK. *P<0.05 versus vehicle (n=6 for each group).

Figure 5.

GQ5 inhibits the transcription activity of phosphorylated Smad3. (A) NRK52E cells are preincubated with or without GQ5 (2.5 μM) for 1 hour before treatment with TGF-β1 (10 ng/ml) for 2 hour. Immunofluorescence staining reveals that GQ5 treatment inhibits TGF-β1–induced nuclei translocation of Smad3, Smad2, and Smad4. (B) NRK52E cells are cotransfected with p(CACA)-luc plasmid and PGL3, followed by TGF-β1 (10 ng/ml) stimulation for 24 hours in the absence or presence of indicated doses of GQ5. Relative luciferase activity is presented. (C and D) NRK52E cells (C) and NRK49F cells (D) are preincubated with or without indicated doses of GQ5 for 1 hour before treatment with TGF-β1 (10 ng/ml) for 36 hours. Real-time PCR is performed to examine the mRNA level of α-SMA, collagen I, and fibronectin. (E and F) NRK52E cells (E) and NRK49F cells (F) are preincubated with or without indicated doses of GQ5 for 1 hour before treatment with TGF-β1 (10 ng/ml) for 48 hours. Western blot is performed to examine the protein expression of α-SMA, collagen I, and fibronectin. Data are expressed as the mean±SD of three independent experiments. P<0.05 in GQ5-treated cells in B–F (ANOVA). *P<0.05 versus untreated cells. DAPI, 4′,6-diamidino-2-phenylindole; Ctrl, control. Original magnification, ×800 in A.

Figure 6.

GQ5 selectively blocks the interaction between TβRI and Smad3 in vitro. NRK52E cells are preincubated with or without GQ5 (2.5 μM) for 1 hour before treatment with TGF-β1 (10 ng/ml) for 1 hour. Cell lysates are collected for immunoprecipitation. (A) Cell lysates are immunoprecipitated with anti-TβRI, followed by immunoblotting using antibodies against Smad3, Smad2, TβRII, and TβRI. (B) Cell lysates are immunoprecipitated with anti-Smad3, followed by immunoblotting using antibodies against Smad3, TβRII, and TβRI. (C) Cell lysates are immunoprecipitated with anti-Smad2, followed by immunoblotting using antibodies against Smad2, TβRII, and TβRI. Data are expressed as the mean±SD of three independent experiments. *P<0.05 versus GQ5-untreated cells under TGF-β1 stimulation. IP, immuno-precipitation; IB, immuno-blotting.

Figure 7.

GQ5 selectively blocks the interaction between TβRI and Smad3 in UUO. Rats receiving daily intraperitoneal injection of vehicle or GQ5 (40 mg/kg per day) are euthanized 14 days after UUO. (A) Kidney homogenates are immunoprecipitated with anti-TβRI, followed by immunoblotting using antibodies against Smad3, Smad2, TβRII, and TβRI. (B) Kidney homogenates are immunoprecipitated with anti-Smad3, followed by immunoblotting using antibodies against Smad3, TβRII, and TβRI. (C) Kidney homogenates are immunoprecipitated with anti-Smad2, followed by immunoblotting using antibodies against Smad2, TβRII, and TβRI. *P<0.05 versus vehicle-treated UUO (n=6 for each group). IP, immuno-precipitation; IB, immuno-blotting.

Figure 8.

GQ5 selectively blocks the interaction between SARA and Smad3. NRK52E cells are preincubated with or without GQ5 (2.5 μM) for 1 hour before treatment with TGF-β1 (10 ng/ml) for 30 minutes. Cell lysates are collected for immunoprecipitation. (A) Cell lysates are immunoprecipitated with anti-SARA, followed by immunoblotting using antibodies against Smad3, Smad2, TβRI, and SARA. (B) Cell lysates are immunoprecipitated with anti-Smad3, followed by immunoblotting using antibodies against SARA and Smad3. (C) Cell lysates are immunoprecipitated with anti-Smad2, followed by immunoblotting using antibodies against SARA and Smad2. Data are expressed as the mean±SD of three independent experiments. *P<0.05 versus GQ5-untreated cells under TGF-β1 stimulation. IP, immuno-precipitation; IB, immuno-blotting.