Epoxyeicosatrienoic acid administration or soluble epoxide hydrolase inhibition attenuates renal fibrogenesis in obstructive nephropathy

Abstract

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites with biological effects, including antiapoptotic, anti-inflammatory, and antifibrotic functions. Soluble epoxide hydrolase (sEH)-mediated hydrolysis of EETs to dihydroxyeicosatrienoic acids (DHETs) attenuates these effects. Recent studies have demonstrated that inhibition of sEH prevents renal tubulointerstitial fibrosis and inflammation in the chronic kidney disease model. Given the pathophysiological role of the EET pathway in chronic kidney disease, we investigated if administration of EET regioisomers and/or sEH inhibition will promote antifibrotic and renoprotective effects in renal fibrosis following unilateral ureteral obstruction (UUO). EETs administration abolished tubulointerstitial fibrogenesis, as demonstrated by reduced fibroblast activation and collagen deposition after UUO. The inflammatory response was prevented as demonstrated by decreased neutrophil and macrophage infiltration and expression of cytokines in EET-administered UUO kidneys. EET administration and/or sEH inhibition significantly reduced M1 macrophage markers, whereas M2 macrophage markers were highly upregulated. Furthermore, UUO-induced oxidative stress, tubular injury, and apoptosis were all downregulated following EET administration. Combined EET administration and sEH inhibition, however, had no additive effect in attenuating inflammation and renal interstitial fibrogenesis after UUO. Taken together, our findings provide a mechanistic understanding of how EETs prevent kidney fibrogenesis during obstructive nephropathy and suggest EET treatment as a potential therapeutic strategy to treat fibrotic diseases.NEW & NOTEWORTHY Epoxyeicosatrienoic acids (EETs) are cytochrome P-450-dependent antihypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered renoprotective. We found that EET administration and/or soluble epoxide hydrolase inhibition significantly attenuates oxidative stress, renal cell death, inflammation, macrophage differentiation, and fibrogenesis following unilateral ureteral obstruction. Our findings provide a mechanistic understanding of how EETs prevent kidney fibrogenesis during obstructive nephropathy and suggest that EET treatment may be a potential therapeutic strategy to treat fibrotic diseases.

Keywords: chronic kidney disease; epoxyeicosatrienoic acids; renal fibrogenesis; soluble epoxide hydrolase; unilateral ureteral obstruction.

Graphical abstract

Figure 1.

Effect of 11,12-epoxyeicosatrienoic acid (EET) and 14,15-EET on kidney fibrosis following unilateral ureteral obstruction (UUO). A: male C57BL/6 mice were subjected to either UUO or sham operation and then administered with the combination of 11,12-EET + 14,15-EET (3, 6, 7.5, or 15 µg/kg/day of each, using an osmotic pump) for 7 days. Kidneys were subjected to Western blot analysis using anti-α-smooth muscle actin (α-SMA) antibody. B: mice were subjected to either UUO or sham operation and then administered with a single treatment of either 11,12-EET or 14,15-EET (15 µg/kg/day). The kidneys were subjected to Western blot analysis using anti-α-SMA antibody. GAPDH was used as a loading control. Band density was measured using ImageJ software. Data are presented as means ± SD; n = 4. ***P < 0.001; ****P < 0.0001. One-way or two-way ANOVA followed by a Tukey’s post hoc multiple comparison test was used to determine significance. NS, not significant; V, vehicle.

Figure 2.

Epoxyeicosatrienoic acid (EET) administration or pharmacological inhibition of soluble epoxide hydrolase attenuates renal interstitial fibrogenesis during unilateral ureteral obstruction (UUO). Male C57BL/6 mice were subjected to either UUO or sham operation and then administered with the combination of 11,12-EET + 14,15-EET (15 µg/kg/day, using an osmotic pump) for 7 days. For the pharmacological inhibition of soluble epoxide hydrolase, t-TUCB (0.4 mg/mouse/day) or vehicle was administered by oral gavage beginning 24 h before UUO. The kidneys were harvested at 7 days after the operation. A: kidney sections were subjected to Sirius red staining and immunohistochemical staining using anti-collagen type I (Col I; brown) and anti-α-smooth muscle actin (α-SMA) antibodies. Hematoxylin stain (blue color) was used for counterstaining. Pictures of the cortex were taken. Scale bars = 50 μm. B: kidneys were subjected to Western blot analysis using anti-α-SMA antibody. GAPDH was used as a loading control. Band density was measured using ImageJ software. C: mRNAs were extracted from kidney tissues using RNA extraction solution as described in materials and methods. The mRNA level of transforming growth factor (TGF)-β was measured and normalized using the GAPDH level. Data are presented as means ± SD; n = 6. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way or two-way ANOVA followed by a Tukey’s post hoc multiple comparison test was used to determine significance. t-TUCB, 4-[[trans-4-[[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]amino]cyclohexyl]oxy]benzoic acid.

Figure 3.

Epoxyeicosatrienoic acid (EET) administration or genetic inhibition of soluble epoxide hydrolase attenuates renal interstitial fibrogenesis during unilateral ureteral obstruction (UUO). Male Ephx2^+/+ and Ephx2^–/– mice were subjected to either UUO or sham operation and then administered with the combination of 11,12-EET + 14,15-EET (15 µg/kg/day, using an osmotic pump) for 7 days. The kidneys were harvested at 7 days after the operation. A: kidney sections were subjected to Sirius red staining (red) and immunohistochemical staining using anti-collagen type I (Col I; brown) and anti-α-smooth muscle actin (α-SMA; brown) antibodies. Hematoxylin stain (blue color) was used for counterstaining. Pictures of the cortex were taken. Scale bars = 50 μm. B: kidneys were subjected to Western blot analysis using anti-α-SMA antibody. GAPDH was used as a loading control. Band density was measured using ImageJ software. C: the mRNA level of transforming growth factor (TGF)-β was measured in the kidneys. mRNA levels were normalized by GAPDH levels. Data are presented as means ± SD; n = 6. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way or two-way ANOVA followed by a Tukey’s post hoc multiple comparison test was used to determine significance. Veh, vehicle; WT, wild type.

Figure 4.

Epoxyeicosatrienoic acid (EET) administration or soluble epoxide hydrolase inhibition reduces renal interstitial inflammation during unilateral ureteral obstruction (UUO). Male C57BL/6 mice were subjected to either UUO or sham operation and then administered with the combination of 11,12-EET + 14,15-EET (15 µg/kg/day, using an osmotic pump) for 7 days. A and B: for the pharmacological inhibition of soluble epoxide hydrolase, t-TUCB (0.4 mg/mouse/day) or vehicle (Veh) was administered by oral gavage beginning 24 h before UUO. The kidneys were harvested at 7 days after the operation. Kidney sections were stained by immunohistochemical staining using anti-F4/80 (brown; A) antibody and antipolymorphonuclear neutrophil (PMN; red; B) antibodies. C and D: Ephx2^+/+ and Ephx2^–/– mice were subjected to UUO or sham operation and then administered with the combination of EETs. Kidney sections were stained by immunohistochemical staining using anti-F4/80 (brown; C) and anti-PMN (red; D) antibodies. Hematoxylin and DAPI stain (both blue) were used for counterstaining. Pictures of the cortex were taken. Scale bars = 50 μm. Data are presented as means ± SD; n = 6. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way or two-way ANOVA followed by a Tukey’s post hoc multiple comparison test was used to determine significance. t-TUCB, 4-[[trans-4-[[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]amino]cyclohexyl]oxy]benzoic acid.

Figure 5.

Epoxyeicosatrienoic acid (EET) regulate macrophage polarization in the kidney during unilateral ureteral obstruction (UUO). Male C57BL/6 mice were subjected to either UUO or sham operation and then administered with the combination of 11,12-EET + 14,15-EET (15 µg/kg/day, using an osmotic pump) for 7 days. A–E: for the pharmacological inhibition of soluble epoxide hydrolase, t-TUCB (0.4 mg/mouse/day) or vehicle (Veh) was administered by oral gavage beginning 24 h before UUO. mRNA levels of inducible nitric oxide synthase (iNOS; A), interleukin (IL)-1β (B), tumor necrosis factor (TNF)-α (C), arginase-1 (Arg-1; D), and IL-10 (E) were measured in the kidneys. F–J: Ephx2^+/+ and Ephx2^–/– mice were subjected to UUO or sham operation and then administered with the combination of EETs. mRNA levels of iNOS (F), IL-1β (G), TNF-α (H), Arg-1 (I), and IL-10 (J) were measured in the kidneys. mRNA levels were normalized by GAPDH levels. Data are presented as means ± SD; n = 6. **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way or two-way ANOVA followed by a Tukey’s post hoc multiple comparison test was used to determine significance. t-TUCB, 4-[[trans-4-[[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]amino]cyclohexyl]oxy]benzoic acid.

Figure 6.

Epoxyeicosatrienoic acid (EET) administration or soluble epoxide hydrolase inhibition reduces macrophage migration and M1/M2 polarization. Raw 264.7 cells were incubated with 1 µM of t-TUCB and/or 1 µM of 11,12-EET and 14,15-EET in the absence or presence of 10 μg/mL of transforming growth factor (TGF)-β for 18 h. A: representative images of bright-field microscopy of hematoxylin and eosin-stained migrated cells. Macrophages were stained pink. Small circles are pores of the membrane filters. The graph summarizes the number of migrated macrophages. Scale bars = 200 μm. mRNA levels of inducible nitric oxide synthase (iNOS; B), interleukin (IL)-1β (C), arginase-1 (Arg-1; D), and IL-10 (E) were measured in the kidneys. mRNA levels were normalized by GAPDH levels. Data are presented as means ± SD; n = 4. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way or two-way ANOVA followed by a Tukey’s post hoc multiple comparison test was used to determine significance. Con, control; t-TUCB, 4-[[trans-4-[[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]amino]cyclohexyl]oxy]benzoic acid; Veh, vehicle.

Figure 7.

Epoxyeicosatrienoic acid (EET) administration or pharmacological or genetic inhibition of soluble epoxide hydrolase suppresses tubular cell damages and cell death during unilateral ureteral obstruction (UUO). Male C57BL/6 mice were subjected to either UUO or sham operation and then administered with the combination of 11,12-EET + 14,15-EET (15 µg/kg/day, using an osmotic pump) for 7 days. The kidneys were harvested at 7 days after the operation. A and B: for the pharmacological inhibition of soluble epoxide hydrolase, t-TUCB (0.4 mg/mouse/day) or vehicle (Veh) was administered by oral gavage beginning 24 h before UUO. A: kidney damage was evaluated by periodic acid-Schiff (PAS) staining and damage scoring. B: kidney sections were stained using a TUNEL assay kit and aquaporin-1 (AQP1) antibody (red). TUNEL-positive cells (green) were counted by microscopy. C and D: Ephx2^+/+ and Ephx2^–/– mice were subjected to either UUO or sham operation and then administered with the combination of 11,12-EET + 14,15-EET. C: kidney damage was evaluated by PAS staining and damage scoring. D: kidney sections were stained using a TUNEL assay kit and AQP1 antibody (red). Hematoxylin and DAPI stain (both blue) were used for counterstaining. Pictures of the cortex were taken. Scale bars = 50 μm. Data are presented as means ± SD; n = 6. **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way or two-way ANOVA followed by a Tukey’s post hoc multiple comparison test was used to determine significance. t-TUCB, 4-[[trans-4-[[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]amino]cyclohexyl]oxy]benzoic acid.

Figure 8.

Epoxyeicosatrienoic acid (EET) administration or pharmacological or genetic inhibition of soluble epoxide hydrolase (sEH) mitigates unilateral ureteral obstruction (UUO)-induced oxidative stress. Male C57BL/6 mice were subjected to either UUO or sham operation and then administered with the combination of 11,12-EET + 14,15-EET (E; 15 µg/kg/day, using an osmotic pump) for 7 days. Kidneys were harvested at 7 days after the operation. A–C: for the pharmacological inhibition of soluble epoxide hydrolase, t-TUCB (T; 0.4 mg/mouse/day) or vehicle (V) was administered by oral gavage beginning 24 h before UUO. A and B: the 4-hydroxynonenal (4-HNE) level was evaluated by Western blot analysis using anti-4-HNE antibody. GAPDH was used as a loading control. Band density was measured using ImageJ software. C: lipid peroxidation as indicated by the lipid hydroperoxide level in kidneys using a lipid hydroperoxide assay kit. C and D: Ephx2^+/+ and Ephx2^–/– mice were subjected to either UUO or sham operation and then administered with the combination of 11,12-EET + 14,15-EET. D and E: the 4-HNE level was evaluated by Western blot analysis using anti-4-HNE antibody. F: lipid peroxidation as indicated by the lipid hydroperoxide level in kidneys. Data are presented as means ± SD; n = 4–6. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way or two-way ANOVA followed by a Tukey’s post hoc multiple comparison test was used to determine significance. TUCB, 4-[[trans-4-[[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]amino]cyclohexyl]oxy]benzoic acid.