Male C57BL/6 mice (20–22 g, 7–8 weeks old) were purchased from Orient Company (Seoul, Korea). All mice were raised in a specific pathogen-free animal facility. All experiments were performed under the approval of the Institutional Animal Care and Use Committee of the Clinical Research Institute at Seoul National University Hospital in accordance with the National Research Council ‘Guidelines for the Care and Use of Laboratory Animals’ [9]. The adamantyl alkyl urea-based sEH inhibitor 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) was synthesized by one of the co-authors of this study as described previously [8,10]. AUDA was dissolved in (2-hydroxypropyl)-β-cyclodextrin (cyclodextrin; Sigma Chemical Co., St Louis, MO, USA) at 45 mg/mL.

Unilateral ureteral obstruction (UUO) was used as a model of kidney fibrosis in C57BL/6 mice. Mice were anesthetized with ketamine (100 mg/kg body weight) and pentobarbital sodium (50 mg/kg body weight, Nembutal; Abbott, Wiesbaden, Germany). After left-flank incisions, UUO was achieved by ligating the left ureter with 4-0 silk at two points and cutting between the ligatures. Mice were placed on a heating pad (40°C) throughout the procedure. Sham-operated mice underwent identical surgical procedures except for ligation of the ureter. The sEH inhibitor AUDA (8 mg/kg/day) was continuously administered via an Alzet^® implantable subcutaneous mini-osmotic pump (DURECT Co., Cupertino, CA, USA) for 1 or 2 weeks. The dose of AUDA was selected based on data from published studies [11,12]. Mice were sacrificed at the indicated times after induction of UUO. Blood pressure was measured before sacrifice using a non-invasive system (Kent Scientific Co., Chicago, IL, USA). Five-to-six mice were used for each group, and three independent experiments were performed for each procedure.

Mice were sacrificed 1 or 2 weeks after UUO, and kidneys were harvested after exsanguination. Tissue samples were fixed with 10% buffered formalin, followed by paraffin embedding. Paraffin sections (4 μm) were then stained with periodic acid-Schiff reagent and Masson’s trichrome staining. Tubulointerstitial lesion indexes were assessed semi-quantitatively from Masson’s trichrome-stained paraffin sections at 100× magnification [13]. A total of 10 fields per kidney were examined by a kidney histologist blinded to the treatment groups, and lesions were graded from 0 to 3 (0, no changes; 1, changes affecting < 25% of the section; 2, changes affecting 25–50% of the section; and 3, changes affecting 50–100% of the section), according to the area with tubulointerstitial lesions (tubular atrophy, casts, interstitial inflammation, and fibrosis). The score index for each mouse was expressed as a mean value of all scores obtained.

Human vascular endothelial cells (HUVECs; Cambrex, Walkersville, MD, USA) were cultured for four to six passages in EGM-2 MV medium (Clonetics Co., San Diego, CA, USA) supplemented with 10% fetal bovine serum, 1 mg/mL hydrocortisone, 12 mg/mL bovine brain extract, 50 mg/mL gentamycin, 50 ng/mL amphotericin B, and 10 ng/mL epidermal growth factor. After 3 days of culture, cells were detached from dishes by the addition of 3 mM ethylenediaminetetraacetic acid solution and a minimal amount of trypsin. Cells (2 × 10⁵/well) were then placed in 8-well chamber slides with serum-free medium for 24 hours and washed twice with phosphate-buffered saline (PBS). Next, recombinant transforming growth factor (rTGF)-β2 (R&D Systems, Minneapolis, MN, USA) was added at a final concentration of 10 ng/mL, except where otherwise indicated. Cells were then incubated with or without AUDA (3 or 6 μM) for 48 hours. Primary human tubular epithelial cells (hTECs) were isolated from unaffected specimens of surgically resected kidneys from patients diagnosed with kidney cell carcinoma [14]. hTECs were cultured for four to six passages in EGM-2 MV medium under the same conditions used for HUVECs.

Confocal microscopy was performed using an LSM 510 meta laser confocal microscope (Carl Zeiss, Jena, Germany). For immunofluorescence studies, kidneys were snap-frozen in optimal cutting temperature embedding medium (Miles Inc., Elkhart, IN, USA), cooled to −80°C, and cut into 5 μm sections using a cryostat (Leica, Heidelberger, Germany). Frozen sections were fixed for 10 minutes in cold acetone. Intrarenal expression levels of fibroblast-specific protein 1 (FSP-1; Abcam, Cambridge, UK), α-smooth muscle actin (αSMA, Abcam), and CD31 (Abcam) were measured by confocal microscopy.

Alexa Fluor^® 488-conjugated goat anti-rabbit antibody (Molecular Probes, Eugene, OR, USA), Alexa Fluor^® 555-conjugated anti-rat antibody, and Alexa Fluor^® 555-conjugated anti-mouse antibody were used as secondary antibodies. All sections were washed and incubated for an additional 5 minutes with 4′,6-diamidi-no-2-phenylindole for counterstaining. For negative controls, primary antibodies were omitted from the staining procedure.

Immunofluorescence analysis of FSP-1 and von Willebrand factor (vWF; Abcam) was carried out using cytospin preparations, generated by centrifugation of 200 μL of cell suspension for 3 minutes at 1,000 rpm onto conventional glass slides using a Shandon Cytospin III cytocentrifuge (Thermo Shandon Ltd., Cambridge, UK).

Immunofluorescence analysis of HUVECs and hTECs in culture was carried out as follows. Cultured HUVECs and hTECs in 8-well-chamber slides were fixed with 2% paraformaldehyde for 5 minutes and then washed twice with PBS. HUVECs were stained for CD31 and FSP-1, and hTECs were stained for E-cadherin (Abcam) and FSP-1. The slides were then fixed with 2% paraformaldehyde for 5 minutes and washed twice with PBS.

In vivo sEH activity was evaluated by measuring the concentrations of EpOME and DHOME using a validated high-performance liquid chromatography/mass spectrometry/mass spectrometry method as described previously [7,8]. Briefly, a 200 μL plasma sample was spiked with 50 μL of internal standard (200 ng/mL of [±] 12,13 DHOME d4), and 2 mL of diethyl ether was added to each sample for liquid–liquid extraction. Detection was performed using an API-4000 QTRAP (Applied Biosystems, Foster City, CA, USA). Eleven working standards were used: 500, 250, 150, 100, 50, 25, 10, 5, 1, 0.5, and 0.1 ng/mL.

Culture supernatants were collected, and the antioxidant reagent triphenylphosphine was added. Aliquots were kept at −80°C until analysis. EET concentration was measured using a 14,15-EETs/DHET ELISA kit (Detroit R&D, Detroit, MI, USA).

Total RNA was extracted from kidney tissues harvested from mice 1 or 2 weeks after induction of UUO. Cytokine mRNA concentrations were assayed by polymerase chain reaction (PCR). Briefly, total RNA was isolated from kidneys using an RNeasy^® kit (Qiagen GmBH, Hilden, Germany), and 1 μg of total RNA was reverse-transcribed using oligo-d(T) primers and AMV-RT Taq polymerase (Promega, Madison, WI, USA). Real-time PCR was performed using Assay-on-Demand TaqMan^® probes and primers for FSP-1, TGF-β1, TGF-β2, vWF, CD31, VE-cadherin, c-kit, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Applied Biosystems) on an ABI PRISM^® 7500 Sequence Detection System (Applied Biosystems). The levels of mRNA expression for each cytokine were normalized to the level of GAPDH mRNA in the same sample.

EndMT and EMT protein levels were analyzed by immunoblotting. Primary antibodies against FSP-1, αSMA, Collagen-4 (Abcam), CD31 (Santa Cruz Biotechnologies, Santa Cruz CA, USA), E-cadherin (Abcam), and β-actin (Sigma-Aldrich, St. Louis, MO, USA) were used for immunoblotting. Briefly, equal amounts (80 μg) of extracted proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto Immobilon-FL 0.4 μM polyvinylidenedifluoride membranes (Millipore, Bedford, MA, USA). Anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) was used as the secondary antibody. Blots were developed using Super Signal West Pico Chemiluminescent Substrate (Pierce, Woburn, MA, USA).

For quantitative flow-cytometry analysis, intrarenal mononuclear cells were isolated from mouse kidney homogenates using a Stomacher^® 80 Biomaster (Seward Ltd., Worthing, Sussex, UK). Single-cell suspensions were created by passing tissues through a 40 μm cell strainer. Kidneys were resuspended in 36% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and overlaid onto 72% Percoll. After centrifugation for 30 minutes at 1,000 g at 25°C, kidney mononuclear cells were isolated from the interface. Isolated kidney mononuclear cells were incubated with fluorophore-conjugated mouse monoclonal antibodies against CD31 and FSP-1 (Bioss, Woburn, MA, USA). Quantitative fluorescence analysis was performed using a FACSCalibur instrument and CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA).

All data are expressed as mean ± standard error of mean (SEM). Group means were compared using the Mann-Whitney U test, one-way ANOVA followed by Tukey’s post-hoc analysis, or two-way ANOVA followed by Bonferroni post-hoc analysis as appropriate. All statistical analyses were performed using GraphPad Prism^® version 4 (GraphPad Software, Inc., La Jolla, CA, USA). P value less than 0.05 was considered to represent statistically significant differences.

We first quantitated the degree of kidney fibrosis induced by UUO in mice using Masson’s trichrome staining. There was extensive tubulointerstitial fibrosis two weeks after UUO; however, AUDA-treated UUO mice exhibited reduced interstitial fibrosis (Fig. 1A, B). To further test the notion that administration of AUDA reduced kidney fibrosis, we performed immunofluorescence analyses. In UUO mice, we detected FSP-1 and αSMA, a marker of fibrosis, in both interstitial and tubule cells. In some instances, we found co localization of FSP-1 and αSMA with CD31, an endothelial cell marker (Fig. 1C–H). Such co-expression of fibroblastic and endothelial markers was interpreted as an indication that cells were undergoing EndMT. In addition to fibroblast proliferation, we also observed destruction of the peritubular capillary network of the kidney in UUO mice. AUDA treatment significantly reduced the level of FSP-1–positive tubulointerstitial fibrosis. Specifically, the numbers of FSP-1/CD31 double-positive fibroblasts and αSMA/CD31 double-positive fibroblasts were considerably decreased after treatment of UUO mice with AUDA. Furthermore, AUDA treatment preserved the peritubular capillary network. With respect to the mRNA level (as determined by real-time PCR) and protein level (as determined by immunoblotting), we quantitatively confirmed that inhibition of sEH down-regulated the expression of FSP-1 (Fig. 1I, J). Blood pressure, monitored non-invasively, was not significantly affected by AUDA treatment (Supplementary fig. 1).

sEH catalyzes the conversion of EpOME to DHOME. To investigate the in vivo activity of sEH, we measured the plasma concentrations of EpOME and DHOME (n = 6 per group). UUO had no effect on the plasma concentration of either compound. However, 9,10-, and total EpOME concentrations were significantly elevated in response to sEH inhibition by AUDA following UUO (Supplementary fig. 2). No significant changes were observed for DHOME plasma concentrations following treatment with AUDA.

To investigate the mechanism underlying the milder interstitial kidney fibrosis observed in AUDA-treated UUO mice, we quantitated TGF-β expression by real-time PCR (Fig. 2A). This analysis revealed that TGF-β1 and TGF-β2 were upregulated at the mRNA level following UUO in C57BL/6 mice; however, TGF-β expression was significantly suppressed after 2 weeks of AUDA treatment.

In addition, we assessed the expression patterns of molecules associated with activation of endothelial cells, including vWF, CD31, and VE-cadherin (Fig. 2B). The expression of these molecules was upregulated by UUO, although the differences were not significant and were increased further by AUDA administration. c-kit (CD117), a cytokine receptor expressed on the surface of hematopoietic stem cells, was also significantly upregulated by AUDA.

We further confirmed that sEH inhibition reduces End-MT by flow-cytometric and immunofluorescence analyses of cytospin preparations. Flow-cytometric analysis demonstrated that the proportion of FSP-1–positive cells was increased in the UUO group relative to the sham-operated group (Fig. 3A). In addition, treatment of UUO mice with AUDA reduced the proportion of CD31/FSP1 double-positive cells in the FSP-1–positive population. We next sorted CD31 cells from isolated intrarenal mono-nuclear cells obtained from mouse kidney homogenates, generated cytospin preparations, and performed double staining for vWF (red) and FSP-1 (green) (Fig. 3B). Cells positive for both vWF and FSP-1 were more abundant in UUO mice, but inhibition of sEH by AUDA reduced the proportion of vWF/FSP-1 double-positive cells.

Histological examination confirmed that fibrosis was ameliorated by AUDA treatment (Fig. 1A). Specifically, AUDA increased the expression of endothelial markers such as vWF, VE-cadherin, and CD31, but decreased expression of TGF-β and FSP-1 (Fig. 2). However, EndMT, as reflected by co-localization of CD31/FSP-1 and CD31/αSMA, is only one aspect of interstitial fibrosis (Fig. 1C–H).

To evaluate the mechanisms by which AUDA reduces tubulointerstitial fibrosis in addition to EndMT, we next performed in vitro studies. Using an in vitro EndMT model, we found that rTGF-β2 treatment caused HUVECs to assume a fibroblast-like morphology and express a high level of FSP-1 (Fig. 4). AUDA treatment prevented this morphological change and reduced expression of FSP-1. Furthermore, AUDA significantly increased the expression of CD31, while reducing expression of smooth muscle actin and FSP-1.

We repeated similar studies in hTEC to evaluate the direct effects of AUDA on EMT (Fig. 5). rTGF-β2 induced morphological changes in TECs, decreased expression of the epithelial cell marker E-cadherin, and increased the level of FSP-1. However, inhibition of sEH did not prevent EMT in cultures that did not contain endothelial cells. To test the possibility that AUDA treatment could reduce EMT of TECs co-cultured with endothelial cells, we grew TECs and HUVECs in separate chambers using the Transwell^™ system (Thermo Fisher Scientific, Roskilde, Denmark) (Fig. 6A). Similar to single cultures of TECs, rTGF-β2 induced EMT in co-cultivated TECs, while AUDA-activated HUVECs prevented EMT in a dose-dependent manner (Fig. 6B–D).

We next measured the concentrations of EETs in culture supernatants (Fig. 6E). AUDA treatment significantly increased the concentration of EET in co-cultures of TECs and HUVECs, but did not affect EET concentration in single cultures of TECs in the absence of endothelial cells. Taken together, these findings suggest that the sEH inhibitor AUDA reduces EMT of TECs by increasing the concentration of EET in vascular endothelial cells.