Omega-3 Fatty Acids Attenuate Renal Fibrosis via AMPK-Mediated Autophagy Flux Activation

Abstract

The unilateral ureteral obstruction (UUO) injury model is well-known to mimic human chronic kidney disease, promoting the rapid onset and development of kidney injury. ω3-poly unsaturated fatty acids (PUFAs) have been observed to protect against tissue injury in many disease models. In this study, we assessed the efficacy of ω3-PUFAs in attenuating UUO injury and investigated their mechanism of action. The immortalized human proximal tubular cells human kidney-2 (HK2) were incubated for 72 h with docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) in various concentrations, in the presence or absence of transforming growth factor (TGF)-β. DHA/EPA reduced the epithelial-mesenchymal transition in the TGF-β-treated HK2 cells by enhancing autophagy flux and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. C57BL/6 mice were divided into four groups and treated as follows: sham (no treatment, n = 5), sham + ω3-PUFAs (n = 5), UUO (n = 10), and UUO + ω3-PUFAs (n = 10). Their kidneys and blood were harvested on the seventh day following UUO injury. The kidneys of the ω3-PUFAs-treated UUO mice showed less oxidative stress, inflammation, and fibrosis compared to those of the untreated UUO mice. Greater autophagic flux, higher amounts of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, Beclin-1, and Atg7, lower amounts of p62, and higher levels of cathepsin D and ATP6E were observed in the kidneys of the omega-3-treated UUO mice compared to those of the control UUO mice. In conclusion, ω3-PUFAs enhanced autophagic activation, leading to a renoprotective response against chronic kidney injury.

Keywords: UUO; autophagy flux; renal fibrosis; ω3-PUFA.

Figure 1

DHA/EPA treatment inhibited the expression of EMT in TGF-β1-treated (10 ng/mL) HK2 cells in a dose-dependent manner. (A,B) Representative Western blot images: DHA/EPA inhibited the expression of α-SMA in a dose-dependent manner in TGF-β1-treated HK2 cells. (C,D) Representative Western blot images: DHA/EPA increased E-cadherin levels in a dose-dependent manner in TGF-β1-treated HK2 cells. * p < 0.05 vs. control HK2 cells, # p < 0.05 vs. TGF-β1-treated HK2 cells in the absence of DHA/EPA. All experiments were performed in triplicate. All values are expressed as the mean ± SD. HK2: human kidney 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; EMT: epithelial–mesenchymal transition; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; TGF: transforming growth factor; SMA: α-smooth muscle actin.

Figure 2

DHA/EPA enhanced autophagic flux via AMPK phosphorylation (A,B). Representative Western blot images: the p-AMPKα/AMPK ratio was decreased in TGF-β-treated (10 ng/mL) HK2 cells compared to control HK2 cells. DHA/EPA increased the p-AMPKα/AMPKα ratio in TGF-beta-treated HK2 cells, in a dose-dependent manner. (C,D) Representative Western blot images: DHA/EPA increased the expression of LC3-II in TGF-beta-treated HK2 cells. in dose-dependent manner. (E,F) Representative Western blot images: DHA/EPA decreased p62 expression in TGF-β1-treated HK2 cells. All experiments were performed in triplicate. All values are expressed as the mean ± SD. * p < 0.05 vs. control HK2 cells; # p < 0.05 vs. TGF-β1-treated HK2 cells in the absence of DHA/EPA. HK2: human kidney 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; EMT: epithelial–mesenchymal transition; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; TGF: transforming growth factor; SMA: α-smooth muscle actin.

Figure 2

DHA/EPA enhanced autophagic flux via AMPK phosphorylation (A,B). Representative Western blot images: the p-AMPKα/AMPK ratio was decreased in TGF-β-treated (10 ng/mL) HK2 cells compared to control HK2 cells. DHA/EPA increased the p-AMPKα/AMPKα ratio in TGF-beta-treated HK2 cells, in a dose-dependent manner. (C,D) Representative Western blot images: DHA/EPA increased the expression of LC3-II in TGF-beta-treated HK2 cells. in dose-dependent manner. (E,F) Representative Western blot images: DHA/EPA decreased p62 expression in TGF-β1-treated HK2 cells. All experiments were performed in triplicate. All values are expressed as the mean ± SD. * p < 0.05 vs. control HK2 cells; # p < 0.05 vs. TGF-β1-treated HK2 cells in the absence of DHA/EPA. HK2: human kidney 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; EMT: epithelial–mesenchymal transition; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; TGF: transforming growth factor; SMA: α-smooth muscle actin.

Figure 3

Anti-inflammatory effects of ω3-PUFAs in kidneys subjected to UUO. (A) Representative photomicrographs (×200) of renal sections stained with H&E and immunostained for 8-OHDG and F4/80. In the H&E images, infiltration of inflammatory cells (yellow arrows) in the interstitial space is evident. (B) Semiquantitative analysis of the interstitial injury score, 8-OHDG-stained area and F4/80-positive cells. (C) mRNA expression of MCP-1 and osteopontin. All experiments for mRNA expression evaluation were performed in triplicate. All values are expressed as the mean ± SD. * p < 0.05 vs. sham, # p < 0.05 vs. UUO-untreated. H&E: hemotoxylin and eosin; 8_OHDG: 8-hydroxy-2′-deoxyguanosine; PUFAs: polyunsaturated fatty acids; UUO: unilateral ureteral obstruction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; OPN: osteopontin; MCP-1: monocyte chemoattractant protein 1; SD: standard deviation.

Figure 3

Anti-inflammatory effects of ω3-PUFAs in kidneys subjected to UUO. (A) Representative photomicrographs (×200) of renal sections stained with H&E and immunostained for 8-OHDG and F4/80. In the H&E images, infiltration of inflammatory cells (yellow arrows) in the interstitial space is evident. (B) Semiquantitative analysis of the interstitial injury score, 8-OHDG-stained area and F4/80-positive cells. (C) mRNA expression of MCP-1 and osteopontin. All experiments for mRNA expression evaluation were performed in triplicate. All values are expressed as the mean ± SD. * p < 0.05 vs. sham, # p < 0.05 vs. UUO-untreated. H&E: hemotoxylin and eosin; 8_OHDG: 8-hydroxy-2′-deoxyguanosine; PUFAs: polyunsaturated fatty acids; UUO: unilateral ureteral obstruction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; OPN: osteopontin; MCP-1: monocyte chemoattractant protein 1; SD: standard deviation.

Figure 4

Anti-fibrosis effects of ω3-PUFA on kidneys subjected to UUO. (A) Representative photomicrographs (×200) of renal sections stained with Masson’s trichrome stain and immunohistochemical staining of α-SMA, collagen IV, and fibronectin. (B) Semiquantitative analysis of the interstitial injury score for α-SMA, collagen IV, and fibronectin-stained areas. (C) Representative Western blot images: expression of fibronectin, collagen IV, and a-SMA in the kidneys of sham and UUO mice treated or not with omega-3 PUFA. All Western blots experiments were performed in triplicate. All values are expressed as the mean ± SD. * p < 0.05 vs. Sham, # p < 0.05 vs. UUO-untreated. MT: Masson’s trichrome stain; COL IV: collagen IV; FN: fibronectin; SMA: smooth muscle actin; PUFAs: polyunsaturated fatty acids; UUO: unilateral ureteral obstruction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

Anti-fibrosis effects of ω3-PUFA on kidneys subjected to UUO. (A) Representative photomicrographs (×200) of renal sections stained with Masson’s trichrome stain and immunohistochemical staining of α-SMA, collagen IV, and fibronectin. (B) Semiquantitative analysis of the interstitial injury score for α-SMA, collagen IV, and fibronectin-stained areas. (C) Representative Western blot images: expression of fibronectin, collagen IV, and a-SMA in the kidneys of sham and UUO mice treated or not with omega-3 PUFA. All Western blots experiments were performed in triplicate. All values are expressed as the mean ± SD. * p < 0.05 vs. Sham, # p < 0.05 vs. UUO-untreated. MT: Masson’s trichrome stain; COL IV: collagen IV; FN: fibronectin; SMA: smooth muscle actin; PUFAs: polyunsaturated fatty acids; UUO: unilateral ureteral obstruction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Autophagy activation in the kidneys of mice subjected to UUO and fed ω3-PUFAs. (A) Representative Western blot images: the levels of Beclin-1, ATG7, LC3-1, LC3-II, p62, ATP6E, and Cathepsin D were assayed. (B) Semiquantitative analysis: expression ratios of Beclin-1, Cathepsin D, LC3-II, p62, ATP6E, and ATG7 to GAPDH, as determined by densitometry. All experiments were performed in triplicate. * p < 0.05 vs. sham, # p < 0.05 vs. UUO-untreated. PUFAs: polyunsaturated fatty acids; UUO: unilateral ureteral obstruction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ATG7: autophagy-related 7; LC3: Microtubule-associated protein 1 light chain 3; ATP6E: V-type proton ATPase subunit E; p62: ubiquitin-binding protein p62.

Figure 6

The effects of ω3-PUFAs on autophagy flux in the kidneys of UUO mice, revealed by immunofluorescence staining of LC3 and LAMP1 in proximal tubules. Comparison of four groups: sham mice, ω3-PUFAs-treated sham mice, UUO mice, and ω3-PUFAs-treated UUO mice. (A) Representative images for LC3 negative control (NC), LC3, LAMP1 (NC), LAMP1, DAPI, and merged images. LC3 staining (fluorescent green cytoplasmic signal corresponding to proximal tubule cells) in proximal tubule cells was increased in UUO mice, compared to sham mice. In UUO mice treated with ω3-PUFAs, LC3 staining of proximal tubule cells was further increased, compared to that in untreated UUO mice. LAMP1 staining (fluorescent red cytoplasmic signal in proximal tubule cells) of proximal tubule cells was increased in the kidneys of UUO mice compared to those of sham mice. LAMP1 staining in proximal tubule cells was decreased in UUO mice treated with ω3-PUFAs compared to untreated UUO mice. The colocalization of LC3 and LAMP, revealed by the orange fluorescence, indicated appropriate autophagy flux (yellow arrows). (B) Quantification of mean fluorescence intensity by ImageJ software bundle with java 8 for Mac OS. The colocalization of LC3 and LAMP1 (orange fluorescence) in the cytoplasm of proximal tubule cells was increased in UUO mice treated with ω3-PUFAs compared to UUO mice. * p < 0.05 vs. sham, # p < 0.05 vs. UUO-untreated. Original magnification, 400×. Scale bar = 20 μm. PUFAs: polyunsaturated fatty acids; UUO: unilateral ureteral obstruction; LC3: Microtubule-associated protein 1 light chain 3; LAMP: lysosomal associated membrane protein.

Figure 6

The effects of ω3-PUFAs on autophagy flux in the kidneys of UUO mice, revealed by immunofluorescence staining of LC3 and LAMP1 in proximal tubules. Comparison of four groups: sham mice, ω3-PUFAs-treated sham mice, UUO mice, and ω3-PUFAs-treated UUO mice. (A) Representative images for LC3 negative control (NC), LC3, LAMP1 (NC), LAMP1, DAPI, and merged images. LC3 staining (fluorescent green cytoplasmic signal corresponding to proximal tubule cells) in proximal tubule cells was increased in UUO mice, compared to sham mice. In UUO mice treated with ω3-PUFAs, LC3 staining of proximal tubule cells was further increased, compared to that in untreated UUO mice. LAMP1 staining (fluorescent red cytoplasmic signal in proximal tubule cells) of proximal tubule cells was increased in the kidneys of UUO mice compared to those of sham mice. LAMP1 staining in proximal tubule cells was decreased in UUO mice treated with ω3-PUFAs compared to untreated UUO mice. The colocalization of LC3 and LAMP, revealed by the orange fluorescence, indicated appropriate autophagy flux (yellow arrows). (B) Quantification of mean fluorescence intensity by ImageJ software bundle with java 8 for Mac OS. The colocalization of LC3 and LAMP1 (orange fluorescence) in the cytoplasm of proximal tubule cells was increased in UUO mice treated with ω3-PUFAs compared to UUO mice. * p < 0.05 vs. sham, # p < 0.05 vs. UUO-untreated. Original magnification, 400×. Scale bar = 20 μm. PUFAs: polyunsaturated fatty acids; UUO: unilateral ureteral obstruction; LC3: Microtubule-associated protein 1 light chain 3; LAMP: lysosomal associated membrane protein.