SRT1720 retards renal fibrosis via inhibition of HIF1A/GLUT1 in diabetic nephropathy

in Journal of Endocrinology
Correspondence should be addressed to Y Shi: yonghongshi@163.com

*(W Han, C Wang and Z Yang contributed equally to this work)

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Renal fibrosis is the major pathological characteristic of diabetic nephropathy (DN). Reportedly, increased SIRT1 expression played a renal protective role in animal models of DN. This study was designed to elucidate the molecular mechanisms underlying the protective effects of SRT1720, an SIRT1 activator, against diabetes-induced renal fibrosis. Type 2 diabetic mice (db/db) were treated with SRT1720 (50 mg/kg/day) by gavage for 10 weeks. Renal proximal tubular epithelial cells (HK-2 cells) were treated with high glucose (HG, 30 mM) in the presence or absence of SRT1720 (2.5 µM) for 48 h. We observed that impaired SIRT1 expression and activity were restored by SRT1720 administration in db/db mice as well as in HG-treated HK-2 cells. Moreover, SRT1720 administration improved the renal function, attenuated glomerular hypertrophy, mesangial expansion, glomerulosclerosis and interstitial fibrosis and inhibited TGFB1 and CTGF expressions and nuclear factor κB (NF-KB) activation in db/db mice. Similarly, HG-induced epithelial-to-mesenchymal transformation (EMT) and collagen IV and fibronectin expressions were inhibited in SRT1720-treated HK-2 cells. Mechanistic studies demonstrated that SRT1720 suppressed HIF1A, GLUT1 and SNAIL expressions both in vivo and in vitro. Furthermore, HIF1A or GLUT1 knockdown effectively abrogated HG-induced EMT and collagen IV and fibronectin expressions in HK-2 cells. These findings suggest that SRT1720 prevented diabetes-induced renal fibrosis via the SIRT1/HIF1A/GLUT1/SNAIL pathway.

 

      Society for Endocrinology

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    Effect of SRT1720 on SIRT1 expression and activity in db/db mice. (A) Immunohistochemical staining images of SIRT1 in kidney sections (scale bar, 20 μm). Arrows indicate positive SIRT1staining. (B) SIRT1 expression was assessed by Western blot in renal cortical homogenates. (C) SIRT1activity. Data are expressed as mean ± s.e.m. (n = 6). *P < 0.05 vs db/m; # P < 0.05 vs db/db.

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    Effect of SRT1720 on renal histopathology. (A) Staining of kidney with periodic acid Schiff (PAS) and Masson Trichrome, and immunofluorescence staining for fibronectin and collagen IV (scale bar, 20 μm). NC, immunofluorescence images obtained from three groups without primary antibody as a negative control. Mesangial index (B), tubulointerstitial injury index (C) and semi-quantitative analyses of collagen IV (D) were measured. (E) Fibronectin expression was detected using Western blot. Data are expressed as mean ± s.e.m. (n = 6). *P < 0.05 vs db/m; # P < 0.05 vs db/db.

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    Effect of SRT1720 on renal expression of E-cadherin, α-SMA, HIF1A, GLUT1 and SNAIL in db/db mice. (A) Immunohistochemical staining of E-cadherin and α-SMA in kidney sections (scale bar, 100 μm). (B) Expressions of E-cadherin and α-SMA were assessed using Western blot. (C) Representative kidney sections of HIF1A and SNAIL expression detected by immunohistochemistry staining and GLUT1 expression detected by immunofluorescence staining (scale bar, 20 μm). Arrows indicate positive HIF1A staining. NC, immunohistochemistry images obtained from three group without primary antibody as a negative control. (D) Expressions of HIF1A, GLUT1 and SNAIL in renal cortex was detected using Western blot. Data are expressed as mean ± s.e.m. (n = 6). *P < 0.05 vs db/m; # P < 0.05 vs db/db.

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    Effect of SRT1720 on expression of TGFB1, CTGF, NF-KB p65, NOX4 and 8-OHdG in diabetic kidneys. (A) Immunohistochemical staining of TGFB1, CTGF, NF-KB p65, NOX4 and 8-OHdG in kidney sections (Scale bar, 20 μm). (B) Protein levels of TGFB1 and CTGF were assessed using Western blot. (C) NF-KB p65 expression in nuclear extracts was assessed using Western blot, and the relative intensity of nuclear NF-KB p65 was normalized against that of Histone H3. (D) NOX4 expression was detected using Western blot. (E) Twenty-four-hour urinary 8-OHdG excretion was measured using ELISA. Data are expressed as mean ± s.e.m. (n = 6). *P < 0.05 vs db/m; # P < 0.05 vs db/db.

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    Effects of SRT1720 on HG-induced EMT, fibronectin and collagen IV expressions, and ROS generation in HK-2 cells. (A) Representative images of immunofluorescence staining for SIRT1, E-cadherin and α-SMA in HK-2 cells. (B) Expressions of SIRT1, E-cadherin and α-SMA were detected using Western blot. (C) SIRT1 activity in HK-2 cells. (D) Morphological changes of HK-2 cells were analysed under an inverted microscope. (E) Representative images of immunofluorescence for fibronectin and collagen IV in HK-2 cells. (F) Fibronectin and collagen IV expressions were detected using Western blot. (G) Mitochondrial ROS generation was assessed using the fluorescence probe MitoSOX Red. Note: Ctrl, 5.6 mM glucose plus 24.4 mM mannitol; HG, high glucose (30 mM); HG + SRT, HG + SRT1720 (2.5 μM). Data are expressed as mean ± s.e.m. (n = 6). *P < 0.05 vs Ctrl; # P < 0.05 vs HG.

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    Effect of SRT1720 on expression of HIF1A, GLUT1 and SNAIL in HK-2 cells. (A) Representative images of immunofluorescence staining for HIF1A and GLUT1. NC, immunofluorescence images obtained from three group without primary antibody as a negative control. (B) Representative images of immunochemistry for SNAIL. NC, immunochemistry images obtained from three group without primary antibody as a negative control. (C) Expressions of HIF1A, GLUT1 and SNAIL were assessed by Western blot. Note: Ctrl, 5.6 mM glucose plus 24.4 mM mannitol; HG, high glucose (30 mM); HG + SRT, HG + SRT1720 (2.5 μM). Data are presented as mean ± s.e.m. (n = 6). *P < 0.05 vs Ctrl; # P < 0.05 vs HG.

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    Effects of HIF1A or GLUT1 knockdown on expression of SNAIL, E-cadherin, α-SMA, fibronectin and collagen IV in HG-treated HK-2 cells. HK-2 cells were transfected with HIF1A siRNA or GLUT1 siRNA and treated with HG for 48 h. (A) Protein levels of HIF1A, GLUT1 and SNAIL were detected using Western blot. (B) Protein levels of E-cadherin and α-SMA were detected using Western blot. (C) Protein levels of fibronectin and collagen IV were detected using Western blot. Note: HG, high glucose (30 mM); HG + Scr, HG + scramble; HG + siHIF1A, HG + HIF1A siRNA; HG + siGLUT1, HG + GLUT1 siRNA. Data are expressed as mean ± s.e.m. (n = 6). *P < 0.05 vs HG.

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    Expressions of SIRT1 and HIF1A in kidneys of patients with DN or normal control kidneys were detected using immunohistochemistry staining. Arrows indicate the positive SIRT1 or HIF1A staining. Scale bar, 50 μm. Negative control, immunohistochemistry images obtained from four groups without primary antibody as a negative control.

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