Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Nov;91(11):1273-84.
doi: 10.1007/s00109-013-1086-1. Epub 2013 Sep 27.

Glucosamine hydrochloride exerts a protective effect against unilateral ureteral obstruction-induced renal fibrosis by attenuating TGF-β signaling

Jinah Park  1 , So-Young Lee, Akira Ooshima, Kyung-Min Yang, Jin Muk Kang, Young-Woong Kim, Seong-Jin Kim
Affiliations

Glucosamine hydrochloride exerts a protective effect against unilateral ureteral obstruction-induced renal fibrosis by attenuating TGF-β signaling

Jinah Park et al. J Mol Med (Berl). 2013 Nov.

Abstract

Renal fibrosis is a common consequence of unilateral ureteral obstruction, which provides a useful model to investigate the pathogenesis of obstructive nephropathy and progressive renal fibrosis. Transforming growth factor (TGF-β1) has been recognized as a key mediator in renal fibrosis by stimulating matrix-producing fibrogenic cells and promoting extracellular matrix deposition. Therefore, considerable efforts have been made to regulate TGF-β signaling for antifibrotic therapy. Here, we investigated the mode of action of glucosamine hydrochloride (GS-HCl) on TGF-β1-induced renal fibrosis. In the obstructed kidneys and TGF-β1-treated renal cells, GS-HCl significantly decreased renal expression of α-smooth muscle actin, collagen I, and fibronectin. By investigating the inhibitory mechanism of GS-HCl on renal fibrosis, we found that GS-HCl suppressed TGF-β signaling by inhibiting N-linked glycosylation of the type II TGF-β receptor (TβRII), leading to an inefficient trafficking of TβRII to the membrane surface. Defective N-glycosylation of TβRII further suppressed the TGF-β1-binding to TβRII, thereby decreasing TGF-β signaling. Notably, GS-HCl treatment significantly reduced TGF-β1-induced up-regulation of Smad2/3 phosphorylation and transcriptional activity in vivo and in vitro. Taken together, GS-HCl-mediated regulation of TGF-β signaling exerted an antifibrotic effect, thereby ameliorating renal fibrosis. Our study suggests that GS-HCl would be a promising agent for therapeutic intervention for preventing TGF-β1-induced renal fibrosis in kidney diseases.

Key message: Glucosamine-mediated attenuation of TGF-β signaling ameliorates renal fibrosis in vivo TGF-β1-induced fibrogenic action is reduced by glucosamine in vitro N-glycosylation of the type II TGF-β receptor is suppressed by glucosamine Glucosamine-induced defective N-glycosylation of TβRII decreases TGF-β signaling.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
GS-HCl exerts an antifibrotic effect in the UUO-induced renal fibrosis model. a Different doses of glucosamine hydrochloride were intraperitoneally injected into mice from 7 days prior to unilateral ureteral obstruction (UUO) surgery. Kidney sections from various groups 14 days after UUO were subjected to Masson’s trichrome staining. Semiquantitative assessment of renal fibrosis was performed with scores using a scale of 0 to 3. Data are the mean ± SEM of four random fields of three paraffin sections prepared from each kidney (n = 5 in each group). * P < 0.05 versus PBS. b Representative photographs of the Masson’s trichrome-stained sham, UUO, and GS-HCl-administered UUO mouse kidneys. Note that 40 and 60 mg/kg of GS-HCl significantly reduce renal fibrotic lesions after obstructive injury. Bar = 50 μm
Fig. 2
Fig. 2
GS-HCl decreases α-SMA, collagen I, and fibronectin expression in the mouse model of UUO-induced renal fibrosis. a–f GS-HCl was daily administered into mice from 7 days prior to UUO. Kidneys were collected for various analyses 14 days after UUO. a Representative RT-PCR and b quantitative real-time RT-PCR show that GS-HCl inhibits α-smooth muscle actin (α-SMA) mRNA expression induced by UUO. * P < 0.05 versus sham control; # P < 0.05 versus UUO + PBS. c Immunofluorescence staining for α-SMA shows that GS-HCl reduces expression of α-SMA protein in the obstructed kidneys. d Representative RT-PCR and e quantitative real-time RT-PCR show that the elevated mRNA expression levels of collagen I and fibronectin by UUO are decreased by GS-HCl administration. *** P < 0.0001, ** P < 0.001 versus sham control; ### P < 0.0001, ## P < 0.001 versus UUO + PBS. f Immunohistochemical staining for fibronectin shows that GS-HCl reduces overdeposition of fibronectin protein in the obstructed kidneys. a, b, d, e Data are the mean ± SEM of three independent measurements. c–f Representative photomicrographs of immunostaining were obtained from evaluating four random fields of each kidney (n = 5 in each group). Bar = 50 μm
Fig. 3
Fig. 3
GS-HCl diminishes TGF-β1-induced fibrogenic responses in renal epithelial cells. a–d HKC-8 cells were treated with 5 mM of GS-HCl for various time intervals. Cells were then incubated with TGF-β1 (3 ng/ml for 16 h) in a serum-free condition. a, b Representative RT-PCR and quantitative RT-PCR data show that GS-HCl inhibits TGF-β1-induced collagen I, fibronectin, and α-SMA mRNA expression, while blocking TGF-β1-mediated suppression of E-cadherin expression. Data are the mean ± SEM of three independent measurements. *** P < 0.0001 versus control without TGF-β1; ### P < 0.0001 versus control with TGF-β1. c Cell lysates were immunoblotted with antibodies to fibronectin, α-SMA, and β-actin. Note that GS-HCl decreases protein expression of TGF-β1-induced fibronectin and α-SMA. d Immunofluorescence staining was performed with fibronectin antibody. DAPI, 4′,6-diamidino-2-phenylindole, was used for nucleus staining (blue). Note that GS-HCl reduces protein expression of TGF-β1-induced fibronectin. Bar = 50 μm
Fig. 4
Fig. 4
GS-HCl reduces TGF-β signaling in the obstructive mouse kidneys and renal epithelial cells. a, b Different doses of GS-HCl were intraperitoneally injected into mice from 7 days prior to UUO surgery. Fourteen days after UUO, a tissue homogenates were immunoblotted with phospho-Smad3 and Smad3. Note that elevated Smad3 phosphorylation induced by UUO was significantly decreased by GS-HCl administration. b Immunohistochemical stainings for phospho-Smad3 in the kidney sections. Note that GS-HCl reduces the Smad3 phosphorylation level elevated by UUO. Representative photomicrographs of immunohistochemical staining were obtained from analyzing four random fields of each kidney (n = 5 in each group). Bar = 50 μm. c, d HKC-8 cells and e mouse primary kidney epithelial cells were treated with GS-HCl with the indicated doses for the indicated periods of time. Cells were then incubated with TGF-β1 (5 ng/ml for 30 min) in a serum-free condition. Cell lysates were immunoblotted with anti-phospho-Smad2/3, anti-Smad2/3, and β-actin. Band intensities representing pSmad2/3 and Smad2/3 expression levels were converted by densitometry using ImageJ software into the ratio of pSmad2/3 to Smad2/3. Note that GS-HCl suppresses phosphorylation of Smad2 and Smad3. f (CAGA)12-luciferase reporter and β-gal were transfected into HKC-8 cells. After GS-HCl (24 h) and tunicamycin (TN; 12 h) treatment, cells were treated with TGF-β1 at 3 ng/ml for 16 h in a serum-free condition. Luciferase activity was normalized with β-gal activity. Note that GS-HCl significantly decreases TGF-β1-induced luciferase activity. *** P < 0.0001, ** P < 0.001 versus control without TGF-β1; ## P < 0.001 versus control with TGF-β1
Fig. 5
Fig. 5
GS-HCl induces defective N-glycosylation of TβRII and reduces TGF-β signaling. a–c HKC-8 cells were transfected with Flag-tagged type II TGF-β receptor (TβRII). After 6 h, GS-HCl (indicated doses for 36 h) or tunicamycin (TN; 1 μg/ml for 12 h) was added to the cell medium. a Cell extracts treated with or without PNGase F were immunoblotted with antibodies against Flag. Note that GS-HCl effectively inhibits N-glycosylation of TβRII. b After GS-HCl or TN treatment, cells were incubated with TGF-β1 (5 ng/ml for 30 min) in a serum-free condition. Cell lysates were immunoblotted with phospho-Smad2/3, Smad2/3, and Flag. Band intensities representing pSmad2/3 and Smad2/3 expression levels were converted by densitometry using ImageJ software into the ratio of pSmad2/3 to Smad2/3. Note that GS-HCl suppresses phosphorylation of Smad2 and Smad3 as well as N-glycosylation of TβRII. c (CAGA)12-luciferase reporter and β-gal were co-transfected with Flag-TβRII into HKC-8 cells. After GS-HCl (24 h) and TN (12 h) treatment, cells were treated with TGF-β1 at 3 ng/ml for 16 h in a serum-free condition. Luciferase activity was normalized with β-gal activity. Note that GS-HCl significantly decreases TGF-β1-induced luciferase activity. *** P < 0.0001, * P < 0.05 versus control without TGF-β1; ### P < 0.0001 versus control with TGF-β1
Fig. 6
Fig. 6
GS-HCl-induced inhibition of TβRII N-glycosylation hinders cell surface transport of TβRII and subsequent TGF-β1-binding. a, b Immunofluorescence staining shows subcellular localization of TβRII (Flag; red), a phalloidin (green), and b protein disulfide isomerase (PDI; green) in HeLa cells. Phalloidin enabled the observation of cellular morphology by staining actin filaments. PDI was used as an ER marker. DAPI, 4′,6-diamidino-2-phenylindole, was used for nucleus staining (blue). Note that GS-HCl interrupts cell surface transport of TβRII and leads to the predominant accumulation of TβRII in the cytosol. Bar = 50 μm. c Representative flow cytometric analysis of receptor density for recombinant human TGF-β1 (rhTGF-β1) at the cell surface. Various amounts of biotinylated TGF-β1 (0–40 ng) were added to 1 × 105 HKC-8 cells treated with or without GS-HCl (5 mM for 36 h). The numbers of biotinylated TGF-β1-bound TβRII were quantified using rhTGF-β1. Note that GS-HCl suppresses TGF-β1 binding to TβRII
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Anderson JW, Nicolosi RJ, Borzelleca JF (2005) Glucosamine effects in humans: a review of effects on glucose metabolism, side effects, safety considerations and efficacy. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 43:187–201. DOI 10.1016/j.fct.2004.11.006 - PubMed
    1. Reginster JY, Deroisy R, Rovati LC, Lee RL, Lejeune E, Bruyere O, Giacovelli G, Henrotin Y, Dacre JE, Gossett C. Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled clinical trial. Lancet. 2001;357:251–256. doi: 10.1016/S0140-6736(00)03610-2. - DOI - PubMed
    1. Wu YL, Kou YR, Ou HL, Chien HY, Chuang KH, Liu HH, Lee TS, Tsai CY, Lu ML. Glucosamine regulation of LPS-mediated inflammation in human bronchial epithelial cells. Eur j pharmacol. 2010;635:219–226. doi: 10.1016/j.ejphar.2010.02.044. - DOI - PubMed
    1. Hwang SY, Shin JH, Hwang JS, Kim SY, Shin JA, Oh ES, Oh S, Kim JB, Lee JK, Han IO. Glucosamine exerts a neuroprotective effect via suppression of inflammation in rat brain ischemia/reperfusion injury. Glia. 2010;58:1881–1892. doi: 10.1002/glia.21058. - DOI - PubMed
    1. Liu Y. Cellular and molecular mechanisms of renal fibrosis. Nat rev Nephrol. 2011;7:684–696. doi: 10.1038/nrneph.2011.149. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources