Regulation of soluble epoxide hydrolase (sEH) activity by adamantyl alkyl urea-based sEH inhibitor (AUDA) in renal ischemia-reperfusion injury (IRI).

<p>A: Plasma epoxyoctadecenoic acid (EpOME) and dihydroxyoctadec-12-enoic acid (DHOME) levels were quantified to investigate the enzyme activity of sEH. B: 9,10-, 12,13-, and total EpOME plasma concentrations were significantly increased in response to AUDA in renal IRI. C: The EpOME/DHOME ratio was significantly increased (*<i>P</i><0.05 compared to sham+vehicle; #<i>P</i><0.05 compared to sham+AUDA; †<i>P</i><0.05 compared to IRI+vehicle).</p

Effects of 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) on renal expression of soluble epoxide hydrolase (sEH) in ischemia-reperfusion injury (IRI) in kidneys.

<p>A: Histological changes were consistent with the functional changes (×200). IRI induced tubular necrosis, consisting of disruption and sloughing of tubular epithelial cells. Arrows indicate necrotic tubules, and asterisks indicate tubular casts. Tubular injury was increased in disease-control mice compared to AUDA-treated mice. B: Expression was quantified by a renal pathologist in a blinded fashion (*<i>P</i><0.05). Scores ranged from 1–5, based on the percentage of tubules affected (1: <10%; 2: 10–25%; 3: 25–50%; 4: 50–75%; 5: >75%). C: sEH was expressed in the endothelium of intraglomerular capillary loops and peritubular capillaries. D and E: Ischemic injury induced the down-regulation of sEH, but AUDA administration had no effect on sEH expression. DAPI was used as counterstaining. <i>EPHX2,</i> gene encoding sEH.</p

Protective effects of soluble epoxide hydrolase (sEH) inhibitor on hypoxic damage via neovascularization.

<p>A: Hypoxia inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), VEGF receptor-2 (KDR), and erythropoietin (EPO) were enhanced by 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) administration. (*<i>P</i><0.05 compared to sham; #<i>P</i><0.05 compared to sham; †<i>P</i><0.05 compared to ischemia-reperfusion injury (IRI) +vehicle). B: Hypoxia induced the down-regulation of sEH in human umbilical vein endothelial cells (HUVECs). Cells were incubated with or without AUDA (10 µM) under hypoxic (1% O₂) or normoxic conditions (20% O₂) for 24 h. Apoptosis of HUVECs was assessed by p53 expression. Hypoxia induced apoptosis in HUVECs, but AUDA treatment reduced apoptosis associated with enhancement of HIF-1α. DAPI was used for counterstaining (magnification, ×400). C and D: c-kit (CD117) expression decreased after IRI, but was amplified by AUDA administration (magnification, ×800). E: AUDA treatment significantly enhanced c-kit expression. Data represent the results of one of three independent experiments (<i>n</i> = 6 per group; †<i>P</i><0.05 compared to IRI+vehicle). F: AUDA treatment increased c-kit and KDR expression levels in HUVECs exposed to hypoxia.</p

Effects of soluble epoxide hydrolase (sEH) inhibitor on the pro-/anti-inflammatory microenvironment in injured kidneys.

<p>A: Proinflammatory cytokines TNF-α and MCP-1 were significantly suppressed, while IL-10 and TGF-β were enhanced by treatment with 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA), as shown by real-time PCR. B: The proinflammatory cytokine IL-6 was decreased and the regulatory cytokines IL-4 and IL-10 were augmented by AUDA, as shown by multiplex cytokine assay. (*<i>P</i><0.05 compared to sham; #<i>P</i><0.05 compared to sham; †<i>P</i><0.05 compared to IRI+vehicle). C: AUDA decreased the infiltration of inflammatory cells (macrophages (F4/80), lymphocytes (CD3), and neutrophils (MPO)) mainly trafficked in the interstitial area. D: AUDA attenuated the infiltration of macrophages/monocytes and T cells expressing CD3, as shown by flow cytometry. F4/80, marker for pan-macrophage; CD44, indicative marker for effector-memory T-cells; CD45, leukocyte common antigen; Gr1, myeloid differentiation antigen.</p

Role of soluble epoxide hydrolase (sEH) activity in ischemia-reperfusion injury (IRI) in kidneys.

<p>A: The adamantyl alkyl urea-based sEH inhibitor, 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) reduced IRI in the kidney. All values are given as means±S.E. (<i>n</i> = 6 per group for each experiment). Data represent one of three independent experiments. Day 0, before bilateral IRI; day 1, 24 h after bilateral IRI; day 2, 48 h after bilateral IRI (two-way ANOVA with Bonferroni post-testing; *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001). B: Administration of AUDA had no effect on blood pressure during the procedure.</p

Multivariate Cox proportional model for renal function progression.

<p>Abbreviations: eGFR, estimated glomerular filtration rate; UPCR, urinary protein to creatinine ratio; cTNFR1, circulating tumor necrosis factor receptor 1; cTNFR2, circulating tumor necrosis factor receptor 2; HR, hazard ratio; CI, confidence interval.</p>a<p>Clinical parameters (Age, sex, hypertension, eGFR, UPCR, pathologic stage, presence of remission, treatment) were examined with cTNFR1.</p>b<p>The effects of cTNFR1 and cTNFR2 were examined separately.</p

Renal expression of TNFRs according to circulating TNFRs.

<p>(A) Immunohistochemical staining of TNFRs expression in paraffin-embedded kidney biopsy sections. TNFR1 is expressed in the glomeruli and tubules in the patients with subnephrotic proteinuria, low cTNFRs levels, and high cTNFRs levels, respectively. TNFR2 is expressed in the tubules but rarely in the glomeruli in the patients with subnephrotic proteinuria, low cTNFRs levels, and high cTNFRs levels, respectively. The intensity of the TNFR2 expression was relatively weaker than that of TNFR1 expression. Original magnification, ×20. (B) Quantitative immunohistochemical analysis of renal TNFRs expression. The quantitative immunohistochemical staining value (QISV, %) was calculated as the integrated optical density divided by the total area occupied by the stained sections in each slide by using computer-assisted quantitative analysis (Qwin3, Leica, Rijswijk, Netherlands). (C) Renal expression of TNFRs in the glomeruli or tubules by performing real-time quantitative reverse transcription-polymerase chain reaction. The Kruskal-Wallis test with Dunn’s method was used. *<i>P</i><0.05.</p

Correlation between the circulating TNFRs levels and autoantibody against phospholipase A₂ receptor (anti-PLA₂R).

<p>(A) The statistically insignificant relationship between Ln cTNFR1 level and anti-PLA₂R. (B) The statistically insignificant relationship between Ln cTNFR2 level and anti-PLA₂R.</p

Proportion of the patients based on severity of pathological findings according to circulating TNFRs levels.

<p>(A) Correlation between the pathological findings and the circulating TNFR1 tertiles. (B) Correlation between the pathological findings and circulating TNFR2 tertiles.</p

Correlation between circulating TNFRs levels and clinical parameters.

<p>Continuous data are expressed as the mean ± SD and categorical data are expressed as numbers (percentage).</p>a<p><i>P</i> value for trend <0.05 (analysis of variance with Scheffe’s multiple comparison test).</p>b<p><i>P</i> value <0.05 compared with TNFRs T1 subgroup. Analysis of variance with Scheffe’s multiple comparison test was used.</p><p>Abbreviations: eGFR, estimated glomerular filtration rate; UPCR, urinary protein to creatinine ratio; cTNFR1, circulating tumor necrosis factor receptor 1; cTNFR2, circulating tumor necrosis factor receptor 2; SD, standard deviation.</p