Am J Physiol Renal Physiol 307: F971–F980, 2014. First published August 27, 2014; doi:10.1152/ajprenal.00256.2014.

Inhibition of soluble epoxide hydrolase prevents renal interstitial fibrosis and inflammation Jinu Kim,1,2,3 John D. Imig,4 Jun Yang,5 Bruce D. Hammock,5 and Babu J. Padanilam1,6 1

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska; 2Department of Anatomy, Jeju National University School of Medicine, Jeju, Republic of Korea; 3Department of Biomedicine and Drug Development, Jeju National University, Jeju, Republic of Korea; 4Department of Pharmacology and Toxicology and Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin; 5Department of Entomology and Comprehensive Cancer Center, University of California, Davis, California; and 6Department of Internal Medicine, Section of Nephrology, University of Nebraska Medical Center, Omaha, Nebraska Submitted 13 May 2014; accepted in final form 15 August 2014

Kim J, Imig JD, Yang J, Hammock BD, Padanilam BJ. Inhibition of soluble epoxide hydrolase prevents renal interstitial fibrosis and inflammation. Am J Physiol Renal Physiol 307: F971–F980, 2014. First published August 27, 2014; doi:10.1152/ajprenal.00256.2014.—The pathophysiological events that lead to renal interstitial fibrogenesis are incompletely understood. Epoxyeicosatrienoic acid (EET), an arachidonic acid metabolite, has anti-inflammatory and profibrinolytic functions. Soluble epoxide hydrolase (sEH) converts EET to less active dihydroxyeicosatrienoic acid. Here, we tested the hypothesis that sEH deficiency would prevent tubulointerstitial fibrosis and inflammation induced by unilateral ureteral obstruction (UUO) in mouse kidneys. The loss of sEH enhanced levels of EET regioisomers and abolished tubulointerstitial fibrosis as demonstrated by reduced collagen deposition and myofibroblast formation at 3 and 10 days after UUO. The inflammatory response was prevented as demonstrated by decreased influx of neutrophil and macrophage, expression of inflammatory cytokines, and chemotactic factors in sEH-deficient UUO kidneys. Pharmacological inhibition of sEH also prevented inflammation and fibrosis after UUO. Next, we delved into the molecular mechanisms piloting the beneficial effects of sEH deficiency in renal fibrosis. UUO upregulated profibrotic factors associated with transforming growth factor (TGF)-␤1/Smad3 signaling, oxidative stress, and NF-␬B activation, and downregulated antifibrotic factors including peroxisome proliferator-activated receptor (PPAR) isoforms, especially PPAR␥, but the loss of sEH prevented these adverse effects in UUO kidneys. Furthermore, administration of PPAR antagonists enhanced myofibroblast formation and activation of Smad3 and NF-␬B p65, effects that were prevented by sEH deficiency in UUO kidneys. These data demonstrate that loss of sEH promotes anti-inflammatory and fibroprotective effects in UUO kidneys via activation of PPAR isoforms and downregulation of NF-␬B, TGF-␤1/Smad3, and inflammatory signaling pathways. Our data suggest the potential use of sEH inhibitors in treating fibrotic diseases. chronic kidney disease; unilateral ureteral obstruction; PPAR isoforms; soluble epoxide hydrolase; tubulointerstitial fibrosis CHRONIC KIDNEY DISEASE (CKD) is progressive, incurable, and ultimately fatal, either because of the consequences of kidney failure or an alarmingly high level of cardiovascular mortality in the CKD patient population (1). Regardless of the disease etiology, including hypertension and diabetes, tubulointerstitial fibrogenesis is the final common pathway in CKD that leads to disease progression and, ultimately, end-stage renal disease

Address for reprint requests and other correspondence: B. J. Padanilam, Dept. of Cellular and Integrative Physiology, 985850 Nebraska Medical Center, Omaha, NE 68198-5850 (e-mail: [email protected]). http://www.ajprenal.org

(ESRD). The past decade has seen great strides in our understanding of renal tubulointerstitial fibrosis, particularly as to how inflammation, fibroblast activation, tubular and microvascular injury contribute to fibrogenesis (3). However, pharmacological interventions based on the functions of many identified pathogenic factors have failed, are inadequate, or at best can slow progression to ESRD, and therefore development of effective drugs to treat renal tubulointerstitial fibrosis remains an elusive goal. The eicosanoid metabolites of the arachidonic acid cascade play important roles in physiology including vasodilatory, anti-inflammatory, and antiapoptotic functions (8, 40). Cytochrome P-450 epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acid (EET), by catalyzing the epoxidation of the olefinic bonds of arachidonic acid, resulting in the production of four regioisomeric EETs: 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET. Soluble epoxide hydrolase (sEH) enzyme converts the EETs to their corresponding less potent diols termed dihydroxyeicosatrienoic acids (DHETs) and therefore the sEH activity is thought to be a major determinant of EET bioavailability (48). Genetic deficiency of sEH increases EET levels in tissues and plasma, potentiates the effects of EETs, and thus elicits antihypertensive and antiinflammatory effect (18). Loss of sEH is also shown to decrease glomerular injury and renal inflammation in rodent models of deoxycorticosterone (DOCA)-salt-induced hypertension (33, 34). Inhibiting the sEH enzyme enhances the beneficial cardiovascular properties of EETs (17). Loss of sEH also reduces renal injury in a CKD model of a type 1 diabetes (11); however, a role for sEH in the pathogenesis of renal interstitial fibrosis is not defined. We hypothesized that diminished availability of EETs could be a potential mechanism for the progression of tubulointerstitial fibrosis, and increasing the bioavailability of EETs is therefore a promising therapeutic strategy for CKD. In this study, we tested the premise that genetic deficiency of sEH may prevent fibrogenesis and inflammation in the unilateral ureteral obstruction (UUO) model of renal interstitial fibrogenesis. MATERIALS AND METHODS

Renal fibrosis model. Male C57BL/6J mice aged 8 to 10 wk were purchased from Jackson Laboratories. Male sEH-knockout (KO) mice were previously reported (34). All mouse experiments were performed in accordance with the animal protocols approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center. UUO was conducted as previously reported

1931-857X/14 Copyright © 2014 the American Physiological Society

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high-power (⫻200 magnification) fields per kidney were used for the counting. Western blot. We performed electrophoresis of protein extracts using tris-glycine buffer systems and subsequent blotting as previously described (24). Membranes were incubated with antibodies against ␣-SMA (Sigma), fibronectin (Cedarlane, Hornby, Ontario, Canada), phosphorylated Smad3 (p-Smad3), phosphorylated NF-␬B p65 (p-p65), poly(ADP-ribose) polymerase 1 (PARP1), caspase-3 (Cell Signaling, Beverly, MA), poly ADP-ribose (PAR; BD Pharmingen, San Jose, CA), intercellular adhesion molecule-1 (ICAM-1; Santa Cruz Biotechnology), TNF-␣ (Abcam), or sEH (Cayman). Peroxidase-conjugated secondary antibodies (Vector Laboratories) were applied, and a chemiluminescence reagent (PerkinElmer, Boston, MA) was used to detect proteins. Anti-␤-actin antibody (Sigma) was used for loading controls on stripped membranes. The bands were quantified using Lab Works analysis software (Ultra-Violet Products, Cambridge, UK). ELISA. The ratios of EETs to DHETs in the kidneys were assessed using EET/DHET ELISA immunoassay kit (Detroit R&D, Detroit, MI). The levels of transforming growth factor (TGF)-␤1, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemotactic protein-1 (MCP-1) in the kidneys were measured using multiplex immunoassay (Millipore, Bedford, MA). The activities of peroxisome proliferator-activated receptor (PPAR) isoforms in the kidneys were measured using PPAR␣, ␤/␦, ␥ transcription factor assay kit (Cayman). The level of lipid hydroperoxide in the kidneys was measured using lipid hydroperoxide assay kit (Cayman). Oxylipin profiling. Oxylipin concentrations in kidney tissues were measured by liquid chromatography/tandem MS analysis as previously described (47). Statistical analyses. ANOVA was used to compare data among groups. Differences between two groups were assessed by two-tailed unpaired Student’s t-test. P values ⬍0.05 were considered statistically significant.

(25). Briefly, the mice were anesthetized with an intraperitoneal injection of a cocktail containing ketamine (200 mg/kg body wt) and xylazine (16 mg/kg body wt). After the left kidney was exposed through the left flank incision, the left ureter was ligated completely near the kidney pelvis using a 5– 0 silk tie. Sham-operated mice underwent the same surgical procedure without the ureter ligation. Glomerulonephritis (GN) was developed as previously reported (35). Briefly, mice were immunized with 1 mg of normal sheep IgG (Jackson ImmunoResearch, West Grove, PA) in Freund’s complete adjuvant (Santa Cruz Biotechnology, Santa Cruz, CA) and injected intravenously with 100 ␮l of sheep anti-rat glomerular basement membrane (GBM) serum (Probetex, San Antonio, TX) 10 days later. Control mice underwent the same procedure without the injection of anti-GBM. For the pharmacological inhibition of sEH, trans-4-[4-(3adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB; 2 or 10 mg/day) (41) or vehicle (10% DMSO in 0.5% methylcellulose) was administered by oral gavage beginning 3 days after UUO. For the pharmacological inhibition of PPAR, a mixture of GW6471 (1 mg·kg body wt⫺1·day⫺1; R&D Systems, Minneapolis, MN) against PPAR␣, GSK0660 (1 mg·kg body wt⫺1·day⫺1; R&D Systems) against PPAR␤/␦, and T0070907 (1.5 mg·kg body wt⫺1·day⫺1; R&D Systems) against PPAR␥ or vehicle (10% DMSO in 0.9% saline) was intraperitoneally injected on the day of UUO surgery. Collagen deposition. Collagen deposition was assessed by both Sirius red staining and hydroxyproline assay as previously described (26). The area of positive Sirius red staining was measured in five randomly chosen high-power (⫻200) fields per kidney using NIH Image J software. Histology. Immunohistochemical staining of the kidneys was performed on paraffin sections as previously described (27). Briefly, 4% paraformaldehyde-fixed kidney sections were rehydrated and labeled with antibodies against ␣-smooth muscle actin (␣-SMA; Sigma, St. Louis, MO), F4/80 (Abcam, Cambridge, MA), or polymorphonuclear neutrophil (PMN; Accurate, Westbury, NY). The sections were then incubated to peroxidase-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA). The quantitative measurement of the immunohistochemical staining was carried out at a suitable magnification (⫻200; 62,500 ␮m2). The respective ␣-SMA- and F4/80positive areas were measured in five randomly chosen fields per kidney using NIH Image J software. The number of PMN-positive cells in five randomly chosen fields per kidney was directly counted under light microscopy (Leica). Periodic acid-Schiff (PAS)-stained sections were used for tubular injury score as described previously (24). Histological damage of tubular injury was scored by percentage of tubules that displayed tubular necrosis, cast formation, and tubular dilation as follows: 0 ⫽ normal, 1 ⫽ ⬍10%, 2 ⫽ 10 to 25%, 3 ⫽ 26 to 50%, 4 ⫽ 51 to 75%, and 5 ⫽ ⬎75%. Ten randomly chosen

RESULTS

Loss of sEH prevents EETs to DHETs conversion in UUO kidneys. To assess the role of sEH during renal interstitial fibrogenesis in vivo, we used a mouse model of obstructive nephropathy. UUO significantly increased the expression of sEH protein in wild-type (WT) mice at 3 and 10 days postinjury compared with that in sham-operated mice (Fig. 1A). No expression of sEH was observed in sEH-deficient mice at any of the time points studied. The ratio of EETs to DHETs was assessed by immunoassay. The ratio of both 11,12-EET/DHET WT

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Fig. 1. Unilateral ureteral obstruction (UUO) increases soluble epoxide hydrolase (sEH) expression and activity. A: expression of kidney sEH in wild-type (WT) or sEH-knockout (KO) mice at 3 or 10 days after sham operation or UUO using Western blot. The bands were quantified using Lab Works analysis software. B: sEH activity presented by ratio of epoxyeicosatrienoic acid (EET) to dihydroxyeicosatrienoic acid (DHET) in the kidneys using EET/DHET ELISA kit (Detroit R&D, Detroit, MI). Error bars represent SD (n ⫽ 5). *P ⬍ 0.05 vs. sham. #P ⬍ 0.05 vs. WT. §P ⬍ 0.05 vs. 3 days. AJP-Renal Physiol • doi:10.1152/ajprenal.00256.2014 • www.ajprenal.org

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lagen deposition as demonstrated by increased Sirius redpositive area (Fig. 2, A and B) and hydroxyproline level (Fig. 2C), whereas the loss of sEH markedly reduced the collagen deposition (Fig. 2, A–C). Tubulointerstitial expression of ␣-SMA, a marker of myofibroblast formation, was induced at 3 and 10 days in WT UUO-induced kidneys but was significantly diminished by the loss of sEH at both time points (Fig. 2, A, D, and E). Three and 10 days after UUO, fibronectin expression level in whole kidneys was also diminished by the loss of sEH (Fig. 2E). We also tested whether loss of sEH prevented renal interstitial fibrosis during GN, another model of renal fibrosis. During GN, collagen deposition in WT kidneys was time-dependently increased as demonstrated by

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and 14,15-EET/DHET decreased in WT compared with that in sEH-KO mice (Ephx2⫺/⫺) kidneys at 10 days after UUO. Levels of sEH-independent oxylipin metabolites showed no significant difference between WT and sEH-KO mice (Supplementary Table S1; the online version of this article contains supplemental data). These data indicate that the increased sEH activity reduced the ratio of EETs to DHETs in WT UUO kidneys consistent with its increased expression levels (Fig. 1B). Loss of sEH prevents renal fibrosis in UUO and GN models. To determine whether sEH activation contributes to interstitial fibrosis induced by UUO, Collagen deposition was assessed by Sirius red staining and hydroxyproline measurement. During UUO, WT kidneys showed a time-dependent increase of col-

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Fig. 2. Loss of sEH prevents interstitial fibrogenesis induced by UUO. A: collagen deposition detected by Sirius red stain and ␣-smooth muscle actin (␣-SMA) expression stained with ␣-SMA monoclonal antibody on kidney sections in WT or sEH-KO mice at 10 days after sham operation or UUO. Arrow heads indicate glomeruli. Scale bars ⫽ 50 ␮m. To demonstrate the specific staining of ␣-SMA in the myofibroblasts in the interstitial area, representative high magnification of the ␣-SMA staining regions from UUO-induced kidneys is shown in the bottom panels. B: percent of Sirius red-positive area on kidney sections. C: collagen content represented by hydroxyproline level in the kidneys. D: percent of ␣-SMA-positive area on the kidney sections. E: profibrotic expression of ␣-SMA and fibronectin in the kidneys evaluated by Western blot analyses. The bands were quantified using Lab Works analysis software. Error bars represent SD (n ⫽ 5). *P ⬍ 0.05 vs. sham. #P ⬍ 0.05 vs. WT. §P ⬍ 0.05 vs. 3 days. AJP-Renal Physiol • doi:10.1152/ajprenal.00256.2014 • www.ajprenal.org

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Fig. 3. Loss of sEH prevents interstitial fibrosis induced by glomerulonephritis (GN). A: collagen deposition detected by Sirius red stain and ␣-SMA expression stained with ␣-SMA monoclonal antibody on kidney sections in WT or sEH-KO mice at 10 days after nonadministration or administration of anti-glomerular basement membrane (GBM) serum. Scale bars ⫽ 50 ␮m. B: percent of Sirius red-positive area on kidney sections. C: collagen content represented by hydroxyproline level in the kidneys. Error bars represent SD (n ⫽ 4). *P ⬍ 0.05 vs. sham. #P ⬍ 0.05 vs. WT. §P ⬍ 0.05 vs. 3 days.

Sirius red-positive area (Fig. 3, A and B) and hydroxyproline level (Fig. 3C), whereas the loss of sEH significantly reduced the collagen deposition at 10 days of GN (Fig. 3, A–C). These data suggest that the loss of sEH has anti-fibrotic effect in different experimental models of CKD. Loss of sEH prevents TGF-␤ expression and its downstream signaling in UUO kidneys. Since myofibroblast differentiation and activation by TGF-␤ signaling are critical events in renal fibrosis (6), we tested whether the TGF-␤1 level is affected by sEH upregulation in WT UUO kidneys. The TGF-␤1 level and its downstream signaling mediator p-Smad3 are significantly reduced in sEH-KO kidneys at 3 and 10 days after UUO, compared with those in WT kidneys (Fig. 4, A and B). These data suggest that sEH upregulation contributes to tubulointerstitial fibrogenesis via TGF-␤1/Smad3 signaling. Loss of sEH diminishes infliltration of neutrophils and macrophages after UUO. To determine whether sEH is implicated in renal inflammation in obstructive nephropathy, we next examined leukocyte influx and proinflammatory response in UUO kidneys. Three and 10 days after UUO, a prominent influx of both PMN-positive neutrophil and F4/80-positive macrophage occurred in WT kidneys, but the loss of sEH significantly prevents inflammatory cell infiltration in UUO kidneys (Fig. 5). Loss of sEH diminishes expression of inflammatory cytokines and chemotactic factors after UUO. To determine whether sEH alters the inflammatory milieu in the UUOinduced kidney, we assessed the levels of various inflammatory

molecules. Loss of sEH decreased levels of keratinocytederived chemokine (KC), MIP-2, and MCP-1 proteins, potent leukocyte chemotactic factors upregulated by UUO (Fig. 6A). Since these chemotactic factors can be transactivated by NF-␬B transcription factor (38, 43, 45), we assessed NF-␬B activation by quantifying p-p65 in UUO kidneys. The level of p-p65 was significantly reduced in sEH-KO kidneys during UUO compared with that in WT kidneys, respectively (Fig. 6B). Expression levels of NF-␬B target gene proteins, TNF-␣ and ICAM-1, were increased in WT kidneys, but were prevented by the loss of sEH during UUO (Fig. 6B). These data suggest that sEH upregulation exacerbates renal inflammation at least partly through an NF-␬B-dependent signaling pathway during interstitial fibrogenesis. Pharmacological inhibition of sEH post-UUO attenuates increase of fibrotic and inflammatory protein expression. To determine whether treatment with tAUCB, a pharmacological inhibitor of sEH, during UUO attenuates renal fibrosis, we measured fibrotic protein expression. Treatment with tAUCB beginning 3 days post-UUO significantly reduced ␣-SMA and fibronectin expression compared with vehicle-treated animals (Fig. 7A). We also assessed the expression of inflammatory proteins in kidneys with or without treatment with tAUCB during UUO. UUO-induced expression of TNF-␣ and ICAM-1 was significantly diminished by pharmacological inhibition of sEH at 10 days after the injury (Fig. 7B). These data suggest that sEH inhibitors may be used as a prophylactic agent to attenuate progression of fibrosis in CKD.

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Fig. 4. Loss of sEH reduces transforming growth factor (TGF)-␤ signaling during UUO. A: kidney TGF-␤1 level in WT or sEH-KO mice after sham operation or UUO was measured by multiplex immunoassay. B: Smad3 phosphorylation (p-Smad3) in the kidneys was analyzed by Western blot. The bands were quantified using Lab Works analysis software. Error bars represent SD (n ⫽ 5). *P ⬍ 0.05 vs. sham. #P ⬍ 0.05 vs. WT. §P ⬍ 0.05 vs. 3 days.

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Fig. 5. Loss of sEH prevents infiltration of neutrophil and macrophage during UUO. The number of polymorphonuclear neutrophil (PMN)-positive neutrophils and the percent of F4/80-positive area on kidney sections in WT or sEH-KO mice after sham operation or UUO was analyzed using immunohistochemistry. The number of neutrophils or macrophages was counted. Error bars represent SD (n ⫽ 5). *P ⬍ 0.05 vs. sham. #P ⬍ 0.05 vs. WT. §P ⬍ 0.05 vs. 3 days.

Loss of sEH diminishes cell death and oxidative stress after UUO. Since inflammation and fibrogenesis can be initiated by cell death (37) and vice versa, we assessed tubular injury and cell death in UUO-subjected kidneys with or without the sEH deficiency. Tubular injury score based on PAS-stained kidney sections was increased at 3 and 10 days after UUO, but the loss of sEH significantly prevented the tubular injury in UUO kidneys (Fig. 8, A and B). The loss of sEH also reduced UUO-induced tubular cell death including both necrosis as demonstrated by PARP1-dependent increased PAR formation and PARP1 expression, and apoptosis demonstrated by cleaved PARP1 and caspase-3 expression (Fig. 8C). To explore

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whether the loss of sEH contributes to decreased oxidative stress during UUO, we next evaluated lipid peroxidation in UUO kidneys in WT and sEH-KO mice. UUO increased lipid peroxidation as represented by the increased level of lipid hydroperoxide in WT kidneys at 3 and 10 days, whereas the loss of sEH ameliorated lipid peroxidation induced by UUO (Fig. 8D), suggesting that sEH upregulation induces oxidative stress in WT UUO-induced kidneys, potentially leading to the progression of tubular injury and cell death. sEH is required for PPAR inactivation during renal fibrosis. PPAR isoforms play a protective role in renal interstitial fibrosis (13, 23). The activity of PPAR isoforms was time-

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Fig. 6. Loss of sEH reduces inflammatory gene expression during UUO. A: levels of chemokine KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemotactic protein-1 (MCP-1) in the kidneys in WT or sEH-KO mice after sham operation or UUO using multiplex immunoassay. B: protein expression of p-p65, TNF-␣, and intercellular adhesion molecule-1 (ICAM-1) in the kidneys using Western blot. The bands were quantified using Lab Works analysis software. Error bars represent SD (n ⫽ 5). *P ⬍ 0.05 vs. sham. #P ⬍ 0.05 vs. WT. §P ⬍ 0.05 vs. 3 days.

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Fig. 7. Pharmacological inhibition of sEH attenuates fibrotic and inflammatory protein expressions induced by UUO. A: profibrotic expression of ␣-SMA and fibronectin in sham or UUO kidneys of mice treated with or without trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB), a sEH inhibitor, evaluated by Western blot analyses. B: proinflammatory expression of TNF-␣ and ICAM-1 in the kidneys evaluated by Western blot analyses. The bands were quantified using Lab Works analysis software. Error bars represent SD (n ⫽ 4). *P ⬍ 0.05 vs. sham. †P ⬍ 0.05 vs. vehicle.

dependently decreased in WT mouse kidneys after UUO, but the loss of sEH prevented the decrease in activity of PPARs after UUO. PPAR␥ activity was reduced by UUO as early as 3 days and persisted at 10 days (Fig. 9), while PPAR␣ and PPAR␤/␥ were reduced only at 10 days post-UUO. The reduced activity of all PPAR isoforms was significantly prevented by the loss of sEH (Fig. 9). To confirm whether the fibrogenic function of sEH is caused by inactivation of PPARs, we administered a mixture of PPAR isoform antagonists to UUO-subjected WT or sEH-KO mice. The administration of the antagonist mixture restored ␣-SMA, p-Smad3, and p-p65 expression at 10 days after UUO in sEH-KO kidneys, but not those in WT kidneys (Fig. 10). These data suggest that the loss of sEH prevents interstitial fibrosis and inflammation via the activation of PPARs. DISCUSSION

Fibrosis is an essential process for survival; however, disregulated fibrosis leads to a variety of deleterious events. Thus, it is no surprise that the pathophysiological events that lead to renal interstitial fibrogenesis are complex and involve several independent and overlapping cellular and molecular signaling pathways. Endothelial cell activation, leukocyte infiltration, tubular cell injury, capillary rarefaction, and pericyte and myofibroblast activation all play important roles in the pathogenesis of renal fibrosis after an inciting insult to the renal parenchyma (2, 7, 30, 44). Traditionally, TGF-␤ signaling has been regarded as a central mediator of tubulointerstitial fibrosis (4, 5). Recently, TGF-␤-independent signaling via other molecules including NF-␬B and angiotensin II is also implicated in renal fibrosis (19, 39). However, the molecular signaling pathways that activate these overlapping pathways to elicit renal fibrogenesis are not defined. In the present study, we tested the effect of the sEH deficiency on the progression of renal failure in mouse UUO

model. Our data indicate that the loss of sEH is beneficial in attenuating the inflammation and interstitial fibrogenesis after UUO. UUO decreased the ratios of EETs to DHETs in WT mouse kidneys that were restored to normal levels by the loss of sEH. The loss of sEH significantly reduced the intrarenal levels of inflammatory cytokines and chemokines, and the infiltration of both neutrophils and macrophages into the renal parenchyma. The loss of sEH by a genetic approach prevented collagen, fibronectin, and ␣-SMA deposition in the UUOinduced kidneys. The loss of sEH also prevented histological damage, oxidative stress, PARP1 and NF-␬B activation, and significantly increased the activities of PPARs, especially the level of PPAR␥. Taken together, increasing the levels of EETs by the sEH deficiency prevents renal interstitial inflammation and fibrogenesis in the mouse UUO model. Our data suggest that the renal sEH activation decreases the EET levels to elicit pathologically relevant inflammatory responses and interstitial fibrogenesis, which are mediated by downregulation of PPAR activity. PPARs are ligand-activated transcription factors. EETs bind to the ligand-binding domain of PPAR␥ with high affinity (32). Several independent reports have demonstrated that PPAR␥ is a negative regulator of profibrotic signaling and blocks matrix deposition and fibrogenesis in diabetic glomerulosclerosis (36) and CCl4-induced liver fibrosis (29). Our data indicate that PPAR levels are downregulated in UUO kidneys. The sEH deletion induces the levels of PPARs and prevents UUOinduced fibrosis. However, the molecular signaling pathways by which PPARs attenuate UUO-induced fibrosis are not well-characterized. A role for PPAR␥ in regulating reactive oxygen species (ROS) levels in the hypothalamus in high fat-fed mice was previously reported (9). The anti-inflammatory effect of PPAR␥ on LPS-induced pulpal inflammation is also due to decreasing ROS levels (28). Our data indicating

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A

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Fig. 8. Loss of sEH suppresses tubular cell damage during interstitial fibrogenesis. A: periodic acid-Schiff (PAS) stain on kidney sections in WT or sEH-KO mice at 10 days after sham operation or UUO. Scale bars ⫽ 50 ␮m. B: tubular injury score represented by PAS stain in the kidneys. C: protein expression of poly ADP-ribose (PAR), poly(ADP-ribose) polymerase 1 (PARP1), cleaved PARP1, and cleaved caspase-3 in the kidneys using Western blot. The bands were quantified using Lab Works analysis software. D: lipid peroxidation indicated by lipid hydroperoxide level in the kidneys using lipid hydroperoxide assay kit. Error bars represent SD (n ⫽ 5). *P ⬍ 0.05 vs. sham. #P ⬍ 0.05 vs. WT. §P ⬍ 0.05 vs. 3 days.

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that the sEH deficiency downregulates oxidative stress in UUO kidneys are consistent with these reports. ROS generated during UUO contributes to oxidative damage to DNA (27). Under conditions of increased DNA damage, PARP1 is hyperactivated and can act as a promoter-specific coactivator of NF-␬B in vivo independent of its enzymatic activity (14, 15). PARP1 binds directly to both subunits of

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NF-␬B (p65 and p50) and promotes NF-␬B-dependent transcription. Our data indicate that the sEH deficiency inhibits PARP1 activation in UUO-induced mice kidneys. Furthermore, the sEH deficiency prevents NF-␬B activation and the inflammatory response. These data concur with earlier reports showing that sEH inhibition attenuated myocardial NF-␬B activation in mice 6 wk after cardiac hypertrophy induced by

#

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Fig. 9. Loss of sEH restricts the reduction of peroxisome proliferator-activated receptors (PPARs) activity induced by UUO. The activity of PPAR isoforms in kidneys in WT or sEH-KO mice after sham operation or UUO was assayed using PPAR␣, ␤/␦, ␥ transcription factor assay kit. Error bars represent SD (n ⫽ 6). *P ⬍ 0.05 vs. sham. #P ⬍ 0.05 vs. WT. §P ⬍ 0.05 vs. 3 days.

Days after UUO

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FIBROGENIC ROLE OF SOLUBLE EPOXIDE HYDROLASE α-SMA AR antagonists PPA

WT

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Fig. 10. PPAR antagonism revives renal fibrosis and inflammation in sEH-KO mice. Expression of ␣-SMA, p-Smad3, and p-p65 in WT and sEH-KO mouse kidneys treated with either vehicle or mixture of PPAR isoform antagonists at 10 days after UUO using Western blot. The bands were quantified using Lab Works analysis software. Error bars represent SD (n ⫽ 4). †P ⬍ 0.05 vs. vehicle.

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thoracic aortic constriction (46). Similarly, NF-␬B activation in the kidney was significantly attenuated in sEH-KO mice with DOCA-salt-induced hypertension (34). As we previously reported (25), in the absence of PARP1, NF-␬B mediated inflammatory mediators TNF-␣ and ICAM-1, and attenuated infiltration of inflammatory cells and the production of cytokines leading to reduced tubulointerstitial lesion and fibrosis in UUO-induced mouse kidneys. In our data, sEH deficiency downregulated TGF-␤1 level in the UUO kidneys. However, the EET-induced signaling pathways that lead to TGF-␤1 downregulation remain undefined. A number of reports implicated a direct role for PPAR␥ in modulation of TGF-␤/Smad3 signaling. The PPAR␥ agonist troglitazone attenuates UUO-induced renal interstitial fibrosis and inflammation through suppression of TGF-␤1 expression (22). Ligand-activated PPAR␥ prevents TGF-␤-induced collagen synthesis via sequestration of essential coactivator p300 from TGF-␤-induced p-Smad complex on the collagen gene promoter (12). It remains to be determined whether EETs may attenuate TGF-␤/Smad3 stimulation via PPAR␥ activation after UUO. Our data are in contrast to a previous reported study in a hypertensive 5/6-Nx model of CKD where sEH inhibition did not delay disease progression but rather resulted in significant increase in glomerulosclerosis, tubulointerstitial damage index, and an increase in albuminuria (21). In previous reports, the antihypertensive effect of sEH inhibition was implicated for decreased renal inflammation and injury in rat and mouse models, like spontaneously hypertensive rats, angiotensin IIinduced hypertension, and DOCA-induced and salt-sensitive hypertension (16, 20, 33). It should be emphasized that inflammation and fibrogenesis observed in our sEH-deficient UUO mouse model are independent of changes in blood pressure. The differences in the results could be due to the model and the strain of mice as the antihypertensive effect in sEH-KO mice was lost after backcrossing on the C57BL/6 background (10, 34). The effect of sEH inhibition on renal protection independently of its effect on lowering blood pressure was previously reported in streptozotocin-induced diabetic mice (11) and in DOCA-salt hypertension (34). Our data are also consistent with a recent report demonstrating the beneficial effects of sEH inhibitors on adverse cardiac remodeling in ischemic cardiomyopathy and pressure-overload hypertrophy (42). On the other hand, in a recent report, pharmacological inhibition of sEH attenuated collagen deposition in angiotensin II-treated heart ventricles, but genetic inhibition of sEH conversely

p-p65 2.5

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aggravated the effect (31) showing differing effects of pharmacological and genetic inhibition of sEH on myocardial fibrosis. In our data, both pharmacological and genetic inhibition had the same preventive effect on collagen deposition in the UUO model of renal fibrosis. The discrepancy may be due to the different sEH pathways activated during fibrogenesis and inflammation in hearts and kidneys. Collectively, these studies as summarized in Fig. 11 demonstrate that increased EETs after the sEH deficiency significantly prevent histological damage, oxidative stress, and increased the levels of PPAR, especially that of PPAR␥. Furthermore, the increased levels of EETs prevented pathologically relevant inflammatory responses including reduced levels of inflammatory cytokines and chemokines, leukocyte infiltration, and

UUO

sEH upregulation • Ratio of EET to DHET ↓ • sEH expression ↑

PPAR inactivation • Transcriptional activity ↓

Inflammation • NF-κB target genes ↑ • Leukocyte influx ↑

Fibrosis • TGF-β1 signaling ↑ • Myofibroblast activation ↑ • Collagen deposition ↑

Fig. 11. Flow diagram of sEH signaling pathway in UUO.

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FIBROGENIC ROLE OF SOLUBLE EPOXIDE HYDROLASE

adverse renal remodeling in the UUO kidneys. Our data demonstrate that EET promotes the anti-inflammatory and fibroprotective effects in UUO kidneys via activation of PPAR isoforms and downregulation of NF-␬B, TGF-␤1/Smad3, and inflammatory signaling pathways. ACKNOWLEDGMENTS We thank Youngsu Cho for technical assistance with the Western blot and immunohistochemistry, and Kelly E. Long and Sherry N. Westphal for mouse care. GRANTS This work is supported by American Heart Association postdoctoral fellowship 13POST16350005 to J. Kim and partially supported by R01 ES002710 and P42 ES004699 to B. D. Hammock. B. J. Padanilam is supported by National Institutes of Health Grants DK-083291 and DK-090332. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: J.K. and B.J.P. conception and design of research; J.K. and J.Y. performed experiments; J.K. analyzed data; J.K., J.D.I., B.D.H., and B.J.P. interpreted results of experiments; J.K. prepared figures; J.K. drafted manuscript; J.K., B.D.H., and B.J.P. edited and revised manuscript; J.K. and B.J.P. approved final version of manuscript. REFERENCES 1. Bakris G, Vassalotti J, Ritz E, Wanner C, Stergiou G, Molitch M, Nesto R, Kaysen GA, Sowers JR. National Kidney Foundation consensus conference on cardiovascular and kidney diseases and diabetes risk: an integrated therapeutic approach to reduce events. Kidney Int 78: 726 –736, 2010. 2. Basile DP, Fredrich K, Chelladurai B, Leonard EC, Parrish AR. Renal ischemia reperfusion inhibits VEGF expression and induces ADAMTS-1, a novel VEGF inhibitor. Am J Physiol Renal Physiol 294: F928 –F936, 2008. 3. Boor P, Ostendorf T, Floege J. Renal fibrosis: novel insights into mechanisms and therapeutic targets. Nat Rev Nephrol 6: 643–656, 2010. 4. Border WA, Okuda S, Languino LR, Sporn MB, Ruoslahti E. Suppression of experimental glomerulonephritis by antiserum against transforming growth factor beta 1. Nature 346: 371–374, 1990. 5. Borges FT, Melo SA, Ozdemir BC, Kato N, Revuelta I, Miller CA, Gattone VH 2nd, LeBleu VS, Kalluri R. TGF-␤1-containing exosomes from injured epithelial cells activate fibroblasts to initiate tissue regenerative responses and fibrosis. J Am Soc Nephrol 24: 385–392, 2013. 6. Bottinger EP. TGF-␤ in renal injury and disease. Semin Nephrol 27: 309 –320, 2007. 7. Campanholle G, Ligresti G, Gharib SA, Duffield JS. Cellular mechanisms of tissue fibrosis. 3. Novel mechanisms of kidney fibrosis. Am J Physiol Cell Physiol 304: C591–C603, 2013. 8. Chen JK, Capdevila J, Harris RC. Cytochrome p450 epoxygenase metabolism of arachidonic acid inhibits apoptosis. Mol Cell Biol 21: 6322–6331, 2001. 9. Diano S, Liu ZW, Jeong JK, Dietrich MO, Ruan HB, Kim E, Suyama S, Kelly K, Gyengesi E, Arbiser JL, Belsham DD, Sarruf DA, Schwartz MW, Bennett AM, Shanabrough M, Mobbs CV, Yang X, Gao XB, Horvath TL. Peroxisome proliferation-associated control of reactive oxygen species sets melanocortin tone and feeding in diet-induced obesity. Nat Med 17: 1121–1127, 2011. 10. Dorrance AM, Rupp N, Pollock DM, Newman JW, Hammock BD, Imig JD. An epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA), reduces ischemic cerebral infarct size in strokeprone spontaneously hypertensive rats. J Cardiovasc Pharmacol 46: 842–848, 2005. 11. Elmarakby AA, Faulkner J, Al-Shabrawey M, Wang MH, Maddipati KR, Imig JD. Deletion of soluble epoxide hydrolase gene improves renal endothelial function and reduces renal inflammation and injury in streptozotocin-induced type 1 diabetes. Am J Physiol Regul Integr Comp Physiol 301: R1307–R1317, 2011.

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