Sirtuin 3 Activation by Honokiol Decreases Unilateral Ureteral Obstruction-Induced Renal Inflammation and Fibrosis via Regulation of Mitochondrial Dynamics and the Renal NF-κB-TGF-β1/Smad Signaling Pathway

Figures

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  Figure 1. Effect of honokiol on unilateral ureteral obstruction (UUO)-induced renal tubular injury and fibrosis. Representative sections of kidneys from sham- and UUO-operated mice treated with vehicle (Veh) or honokiol (HKL) were stained with (A) Periodic acid-Schiff stain (PAS) and (B) Masson’s trichrome (MTC). Scale bar = 100 μm. The bar graph shows semi-quantitative scoring of tubular injury by PAS, area fractions (%) of tubulointerstitial fibrosis, and degree of interstitial collagen deposition stained by MTC in the sham and UUO-operated kidneys. Ten randomly chosen, non-overlapping fields were quantified (n = 10 per group). Data are expressed as mean ± SD. *** p < 0.001 versus Veh or HKL; ### p < 0.001 versus UUO treatment with Veh; Sham, sham-operated mice; UUO, unilateral ureteral obstruction; Veh, vehicle; HKL, honokiol.

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  Figure 2. Effect of honokiol on unilateral ureteral obstruction (UUO)-induced myofibroblast activation and extracellular matrix deposition. (A) Representative sections of kidneys from sham- and UUO-operated mice treated with Vehicle (Veh) or honokiol (HKL) were immunofluorescence-stained with α-smooth muscle actin (α-SMA) (red). The nucleus was stained by DAPI (blue). Scale bar = 50 μm. The bar graph shows area fractions of α-SMA (%) in the sham and UUO kidneys from ten randomly chosen, non-overlapping fields at a magnification of 400× (n = 10 per group). (B) The sections were stained with Picro Sirius Red. Scale bar = 100 μm. The bar chart shows Picro Sirius Red (+) areas (%) in the sham and UUO kidneys from ten randomly chosen, non-overlapping fields at a magnification of 200× (n = 10 per group). (C) Representative Western blot analysis of α-SMA and type I collagen expression in the kidneys from sham- and UUO-operated mice treated with Veh or HKL. The bar graph shows the densitometric quantification presented as the relative ratio of each protein to GAPDH. The relative ratio measured in the kidneys from sham-operated mice treated with Veh is arbitrarily presented as 1. Data are expressed…

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  Figure 3. Effect of honokiol on unilateral ureteral obstruction (UUO)-induced renal inflammation. (A) Representative sections of kidneys from sham- and UUO-operated mice treated with vehicle (Veh) or honokiol (HKL) were immunofluorescence-stained with F4/80 (red). The nucleus was stained with DAPI (blue). Scale bar = 50 μm. The bar graph shows the number of F4/80-positive cells (%) in the sham and UUO kidneys from ten randomly chosen, non-overlapping fields at a magnification of 400× (n = 10 per group). (B) Representative sections of kidneys from sham- and UUO-operated mice treated with Veh or HKL were stained with ICAM-1 and MCP-1. Scale bar = 100 μm. The bar graph shows area fractions (%) of ICAM-1 and MCP-1 in the sham- and UUO-operated kidneys from ten randomly chosen, non-overlapping fields at a magnification of 400× (n = 10 per group). (C) Representative Western blot analysis of ICAM-1 protein expression in the kidneys from sham- and UUO-operated mice treated with Veh or HKL. The bar graph shows the densitometric quantification presented as the relative ratio of each protein to GAPDH. The relative ratio measured in the kidneys from sham-operated mice treated with Veh is arbitrarily…

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  Figure 4. Effect of honokiol on nuclear factor-κB (NF-κB) signaling pathway after ureteral obstruction. (A) Representative sections of kidneys from sham- and unilateral ureteral obstruction (UUO)-operated mice treated with vehicle (Veh) or honokiol (HKL) were stained with p65. Scale bar = 50 μm. The bar graph shows the number of nuclear p65 (+) cells in the sham- and UUO-operated kidneys from ten randomly chosen, non-overlapping fields at a magnification of 400× (n = 10 per group). (B) Representative Western blot analysis of phospho-p65 protein expression in the kidneys from sham- and UUO-operated mice treated with Veh or HKL. The bar graph shows the densitometric quantification presented as the relative ratio of each protein to GAPDH. The relative ratio measured in the kidneys from sham-operated mice treated with Veh is arbitrarily presented as 1. Data are expressed as mean ± SD. *** p < 0.001 versus Veh or HKL; # p < 0.05 and ### p < 0.001 versus UUO treatment with Veh. Sham, sham-operated mice; UUO, unilateral ureteral obstruction; Veh, vehicle; HKL, honokiol; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Figure 5. Effect of honokiol on unilateral ureteral obstruction (UUO)-induced Sirtuin 3 (SIRT3) expression and mitochondrial dynamics. (A) Representative Western blot analysis of SIRT3, dynamic-related protein (DRP)-1, and optic atrophy 1 (OPA)-1 protein expression from sham- and UUO-operated mice treated with vehicle (Veh) or honokiol (HKL). The bar graph shows the densitometric quantification presented as the relative ratio of each protein to GAPDH. The relative ratio measured in the kidneys from sham-operated mice treated with Veh is arbitrarily presented as 1. Data are expressed as mean ± SD. (B) Representative electron micrographs of kidneys from sham- and UUO-operated mice treated with Veh or HKL and their quantification of mitochondrial contour measurements. Scale bar = 2 μm. Data are expressed as mean ± SD of mitochondrial contour measurements from six to eight proximal tubules per mouse (n = 10 per group). *** p < 0.001 versus Veh or HKL; # p < 0.05, ## p < 0.01 and ### p < 0.001 versus UUO treatment with Veh. Sham, sham-operated mice; UUO, unilateral ureteral obstruction; Veh, vehicle; HKL, honokiol; DRP-1, dynamic-related protein-1; OPA-1, optic atrophy 1; Sirtuin 3, SIRT3.

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  Figure 6. Effect of honokiol on TGF-β1-induced fibroblast proliferation and cell migration in NRK-49F cells (A) NRK-49F cells were treated with vehicle (Veh) or transforming growth factor-β1 (TGF-β1, 2 ng/mL), with or without honokiol (HKL) at indicated doses (0.1, 1, and 10 μM). After 24 h of treatment, cell proliferation was measured by XTT assay. Data are expressed as mean ± SD for three independent experiments in triplicates. *** p < 0.001 versus Veh or HKL; ## p < 0.01, ### p < 0.001 versus TGF-β1 (2 ng/mL). (B) Representative phase-contrast images for NRK-49F cells after wound healing assay. The phase-contrast images of migration of NRK-49F cells into a scratch area were obtained after treatment with either Veh or TGF-β1 (2 ng/mL), with or without HKL (10 μM), at 0 or 24 h after wounding. The bar graph shows the average length by which the gap between the NRK-49F cells closed over 0 or 24 h after treatment with Veh, TGF-β1, and/or HKL. *** p < 0.001 versus Veh or HKL; ### p < 0.001 versus TGF-β1. Bar = 200 μm. Veh, vehicle; HKL, honokiol; TGF-β1, transforming growth factor-β1.

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  Figure 7. Effect of honokiol on transforming growth factor-β1 (TGF-β1)-induced fibroblast activation and extracellular matrix protein expression by regulation of the TGF-β1/Smad signaling pathway in NRK-49F cells (A) Representative Western blot for α-SMA, fibronectin, and type I collagen from NRK-49F cells treated with vehicle (Veh) or TGF-β1 (2 ng/mL), with or without honokiol (HKL) at indicated doses (0.1, 1, and 10 μM). Treatment with TGF-β1 (2 ng/mL) for 24 h increased the expression of fibrotic markers. The expression of α-smooth muscle actin (α-SMA), fibronectin, and type I collagen decreased after HKL treatment in a dose-dependent manner. The bar graph shows the densitometric quantification presented as the relative ratio of each protein to GAPDH. Data are presented as mean ± SD. (B) Representative Western blot for phospho-Smad2 and phospho-Smad3 expression from NRK-49F cells treated with vehicle (Veh) or TGF-β1 (2 ng/mL), with or without honokiol (HKL) at indicated doses (0.1, 1, and 10 μM). TGF-β1 (2 ng/mL) treatment for 30 min increased the expression of phospho-Smad2 and phospho-Smad3. Measurement of phospho-Smad2 and phospho-Smad3 protein expression of densitometry is presented…