AMP-activated protein kinase activation ameliorates eicosanoid dysregulation in high-fat-induced kidney disease in mice
2019; Elsevier BV; Volume: 60; Issue: 5 Linguagem: Inglês
10.1194/jlr.m088690
ISSN1539-7262
AutoresAnne‐Émilie Declèves, Anna V. Mathew, Aaron M. Armando, Xianlin Han, Edward A. Dennis, Oswald Quehenberger, Kumar Sharma,
Tópico(s)Alcohol Consumption and Health Effects
ResumoHigh-fat diet (HFD) causes renal lipotoxicity that is ameliorated with AMP-activated protein kinase (AMPK) activation. Although bioactive eicosanoids increase with HFD and are essential in regulation of renal disease, their role in the inflammatory response to HFD-induced kidney disease and their modulation by AMPK activation remain unexplored. In a mouse model, we explored the effects of HFD on eicosanoid synthesis and the role of AMPK activation in ameliorating these changes. We used targeted lipidomic profiling with quantitative MS to determine PUFA and eicosanoid content in kidneys, urine, and renal arterial and venous circulation. HFD increased phospholipase expression as well as the total and free pro-inflammatory arachidonic acid (AA) and anti-inflammatory DHA in kidneys. Consistent with the parent PUFA levels, the AA- and DHA-derived lipoxygenase (LOX), cytochrome P450, and nonenzymatic degradation (NE) metabolites increased in kidneys with HFD, while EPA-derived LOX and NE metabolites decreased. Conversely, treatment with 5-aminoimidazole-4-carboxamide-1-β-D-furanosyl 5′-monophosphate (AICAR), an AMPK activator, reduced the free AA and DHA content and the DHA-derived metabolites in kidney. Interestingly, kidney and circulating AA, AA metabolites, EPA-derived LOX, and NE metabolites are increased with HFD; whereas, DHA metabolites are increased in kidney in contrast to their decreased circulating levels with HFD. Together, these changes showcase HFD-induced pro- and anti-inflammatory eicosanoid dysregulation and highlight the role of AMPK in correcting HFD-induced dysregulated eicosanoid pathways. High-fat diet (HFD) causes renal lipotoxicity that is ameliorated with AMP-activated protein kinase (AMPK) activation. Although bioactive eicosanoids increase with HFD and are essential in regulation of renal disease, their role in the inflammatory response to HFD-induced kidney disease and their modulation by AMPK activation remain unexplored. In a mouse model, we explored the effects of HFD on eicosanoid synthesis and the role of AMPK activation in ameliorating these changes. We used targeted lipidomic profiling with quantitative MS to determine PUFA and eicosanoid content in kidneys, urine, and renal arterial and venous circulation. HFD increased phospholipase expression as well as the total and free pro-inflammatory arachidonic acid (AA) and anti-inflammatory DHA in kidneys. Consistent with the parent PUFA levels, the AA- and DHA-derived lipoxygenase (LOX), cytochrome P450, and nonenzymatic degradation (NE) metabolites increased in kidneys with HFD, while EPA-derived LOX and NE metabolites decreased. Conversely, treatment with 5-aminoimidazole-4-carboxamide-1-β-D-furanosyl 5′-monophosphate (AICAR), an AMPK activator, reduced the free AA and DHA content and the DHA-derived metabolites in kidney. Interestingly, kidney and circulating AA, AA metabolites, EPA-derived LOX, and NE metabolites are increased with HFD; whereas, DHA metabolites are increased in kidney in contrast to their decreased circulating levels with HFD. Together, these changes showcase HFD-induced pro- and anti-inflammatory eicosanoid dysregulation and highlight the role of AMPK in correcting HFD-induced dysregulated eicosanoid pathways. The prevalence of obesity has continued to rise over the past few decades and was 36.5% among US adults between the years 2011 and 2014 (1.Ogden C.L. Carroll M.D. Fryar C.D. Flegal K.M. Prevalence of obesity among adults and youth: United States, 2011–2014.NCHS Data Brief. 2015; 219: 1-8PubMed Google Scholar). Obesity serves as a significant risk factor for both the initiation and progression of kidney disease independent of hypertension and diabetes (2.Mathew A.V. Okada S. Sharma K. Obesity related kidney disease.Curr. Diabetes Rev. 2011; 7: 41-49Crossref PubMed Scopus (79) Google Scholar, 3.Bruce K.D. Byrne C.D. The metabolic syndrome: common origins of a multifactorial disorder.Postgrad. Med. J. 2009; 85: 614-621Crossref PubMed Scopus (119) Google Scholar, 4.de Vries A.P. Ruggenenti P. Ruan X.Z. Praga M. Cruzado J.M. Bajema I.M. D'Agati V.D. Lamb H.J. Pongrac Barlovic D. Hojs R. et al.Fatty kidney: emerging role of ectopic lipid in obesity-related renal disease.Lancet Diabetes Endocrinol. 2014; 2: 417-426Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Excessive intake of calorie-dense lipids leads to organ dysfunction both by direct lipotoxicity and inflammation. In fact, the Western diet enriched in saturated animal fats has been shown to increase albuminuria and cause a faster decline in renal function (5.Lin J. Fung T.T. Hu F.B. Curhan G.C. Association of dietary patterns with albuminuria and kidney function decline in older white women: a subgroup analysis from the Nurses' Health Study.Am. J. Kidney Dis. 2011; 57: 245-254Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). In our previous studies, we established that high-fat diet (HFD)-induced kidney disease is characterized by renal hypertrophy, increased albuminuria, and elevated markers of renal fibrosis and inflammation (6.Declèves A.E. Mathew A.V. Cunard R. Sharma K. AMPK mediates the initiation of kidney disease induced by a high-fat diet.J. Am. Soc. Nephrol. 2011; 22: 1846-1855Crossref PubMed Scopus (176) Google Scholar). These HFD-induced markers of inflammation, oxidative stress, and fibrosis are reversed by AMP-activated protein kinase (AMPK) activation (6.Declèves A.E. Mathew A.V. Cunard R. Sharma K. AMPK mediates the initiation of kidney disease induced by a high-fat diet.J. Am. Soc. Nephrol. 2011; 22: 1846-1855Crossref PubMed Scopus (176) Google Scholar, 7.Declèves A.E. Zolkipli Z. Satriano J. Wang L. Nakayama T. Rogac M. Le T.P. Nortier J.L. Farquhar M.G. Naviaux R.K. et al.Regulation of lipid accumulation by AMK-activated kinase in high fat diet-induced kidney injury.Kidney Int. 2014; 85: 611-623Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). HFD alters the activity of the crucial lipid metabolism enzymes, acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), contributing to lipid accumulation in the kidney (6.Declèves A.E. Mathew A.V. Cunard R. Sharma K. AMPK mediates the initiation of kidney disease induced by a high-fat diet.J. Am. Soc. Nephrol. 2011; 22: 1846-1855Crossref PubMed Scopus (176) Google Scholar). Total cholesterol esters and phosphatidylcholine content in the kidney are elevated, while the FA and triglyceride content is unchanged. Phospholipid accumulation in the proximal tubules is associated with lysosomal dysfunction, stagnant autophagic flux, mitochondrial dysfunction, and inflammasome activation (8.Yamamoto T. Takabatake Y. Takahashi A. Kimura T. Namba T. Matsuda J. Minami S. Kaimori J.Y. Matsui I. Matsusaka T. et al.High-fat diet-induced lysosomal dysfunction and impaired autophagic flux contribute to lipotoxicity in the kidney.J. Am. Soc. Nephrol. 2017; 28: 1534-1551Crossref PubMed Scopus (130) Google Scholar). High-fat feeding for long periods causes recruitment of macrophages, switch to macrophage pro-inflammatory phenotype, and increased inflammatory mediators like TNFα, monocyte chemoattractant protein-1 (MCP-1), IL-6, cyclooxygenase (COX)-2, and IL-1β (9.Börgeson E. Wallenius V. Syed G.H. 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Eicosanoids are oxylipins derived from arachidonic acid (AA) or related PUFAs and are inextricably related to inflammation in the kidney. The primary PUFAs for the n-6 series and n-3 series, linoleic acid (LA) and α-linolenic acid (ALA), respectively, are both derived from the diet. These 18-carbon PUFAs are then metabolized by various desaturase and elongase enzymes in a stepwise fashion. However, both LA and ALA are acted on by the same enzymes, resulting in a competition between the n-3 and n-6 series (14.Nakamura M.T. Nara T.Y. Essential fatty acid synthesis and its regulation in mammals.Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 145-150Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). LA is metabolized through multiple steps to dihomo-γ-linolenic acid (DGLA; 20:3n6) and, ultimately, to AA (20:4n6). On the other hand, ALA is metabolized to EPA (20:5 n-3) and subsequently to DHA (22:6 n-3) (15.Schmitz G. Ecker J. The opposing effects of n-3 and n-6 fatty acids.Prog. Lipid Res. 2008; 47: 147-155Crossref PubMed Scopus (870) Google Scholar). These PUFAs are incorporated into membrane phospholipids and released by phospholipase A2 (PLA2) under the influence of various stimuli. In subsequent reactions, COXs, lipoxygenases (LOXs), and cytochrome P450 (P450) enzymes act on free PUFAs to form eicosanoids. Some eicosanoids can also be formed from PUFAs via nonenzymatic reactions [nonenzymatic degradation (NE)], e.g., isoprostanes. Eicosanoids play an essential role in the regulation of renal physiology and disease by modulating renal blood flow, glomerular filtration rate, autoregulation, tubular glomerular feedback, excretion of renal water and sodium, and release of renin and erythropoietin. HFD feeding causes an increase in circulating eicosanoids. In the kidney, these eicosanoids are produced by all different cell types: mesangial cells, renal microvessels, and tubular cells. This makes it difficult to pinpoint the actual origin of these autacoids without actual profiling of the various compartments. Local production in the kidney will be reflected in the kidney tissue, renal venous compartment, and urine. Recent advances in eicosanoid analysis using highly sensitive MS have enabled us to profile over 150 different eicosanoid metabolites reliably in all tissues, enabling us to systematically profile the changes in the metabolic pathways with HFD and 5-aminoimidazole-4-carboxamide-1-β-D-furanosyl 5′-monophosphate (AICAR) therapy. AMPK is a ubiquitous heterotrimeric kinase that acts as a cellular energy sensor that responds to changes in the intracellular AMP/ATP ratio (16.Carling D. The AMP-activated protein kinase cascade–a unifying system for energy control.Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (957) Google Scholar). AICAR acts as a specific AMPK agonist (17.Corton J.M. Gillespie J.G. Hawley S.A. 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Nephrol. 2011; 22: 1846-1855Crossref PubMed Scopus (176) Google Scholar). Along with lipid accumulation, the markers of inflammation were modulated with AICAR use (7.Declèves A.E. Zolkipli Z. Satriano J. Wang L. Nakayama T. Rogac M. Le T.P. Nortier J.L. Farquhar M.G. Naviaux R.K. et al.Regulation of lipid accumulation by AMK-activated kinase in high fat diet-induced kidney injury.Kidney Int. 2014; 85: 611-623Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). AMPK signaling has been shown to influence the secretory PLA2 expression in vascular smooth muscle cells (19.El Hadri K. Denoyelle C. Ravaux L. Viollet B. Foretz M. Friguet B. Rouis M. Raymondjean M. AMPK signaling involvement for the repression of the IL-1β-induced group IIA secretory phospholipase A2 expression in VSMCs.PLoS One. 2015; 10: e0132498Crossref PubMed Scopus (5) Google Scholar) and control triglyceride content in adipocytes (20.Jiang S. Chen H. Wang Z. Riethoven J.J. Xia Y. Miner J. Fromm M. Activated AMPK and prostaglandins are involved in the response to conjugated linoleic acid and are sufficient to cause lipid reductions in adipocytes.J. Nutr. Biochem. 2011; 22: 656-664Crossref PubMed Scopus (13) Google Scholar). AMPK activation also decreases the formation of 15-LOX metabolites of AA in macrophages (21.Namgaladze D. Snodgrass R.G. Angioni C. Grossmann N. Dehne N. Geisslinger G. Brüne B. AMP-activated protein kinase suppresses arachidonate 15-lipoxygenase expression in interleukin 4-polarized human macrophages.J. Biol. Chem. 2015; 290: 24484-24494Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). While AMPK activation is beneficial in lipid and eicosanoid metabolism in other tissues, the effect of HFD and AMPK activation on eicosanoid pathways in the kidney is unknown. We hypothesized that the high-fat exposure triggers inflammation involving the eicosanoid pathway and that eicosanoid production is ameliorated with AMPK activation. We used a targeted lipidomic platform to systematically investigate the HFD-associated eicosanoid synthesis induced in mice consuming HFD with or without AMPK activation in order to better understand the pathophysiological processes involved in HFD-induced kidney disease. All animal procedures were approved by the Institutional Animal Care and Use Committee of University of California, San Diego. Male 6-week-old C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME) and fed either a standard diet (STD) [5% fat (PUFA, 2.1%; n-6, 1.9%; n-3, 0.2%), 24.5% protein, 40% carbohydrate] or a HFD [60% of total calories from fat (90% lard + 10% soybean oil; PUFA, 16.9%; n-6, 15.1%; n-3, 1.7%), 20% protein, 20% carbohydrate] (D12492; Research Diets, New Brunswick, NJ) for 14 weeks. The mice on the STD were treated with PBS and mice on HFD were treated with AICAR (0.5 mg/g body weight; Toronto Chemicals) or PBS via intraperitoneal injections for 5 days a week for a total of 14 weeks (6.Declèves A.E. Mathew A.V. Cunard R. Sharma K. AMPK mediates the initiation of kidney disease induced by a high-fat diet.J. Am. Soc. Nephrol. 2011; 22: 1846-1855Crossref PubMed Scopus (176) Google Scholar). Mice were placed in metabolic cages for 24 h urine collection before the start of the diets and after 14 weeks. Mice were euthanized after 14 weeks of diet, plasma was collected from the renal vein, and arterial blood was obtained from direct cardiac puncture. After perfusion with PBS, kidneys were snap-frozen in liquid nitrogen for further analysis. Total and free FAs were analyzed by GC-MS as previously described (22.Quehenberger O. Armando A.M. Dennis E.A. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry.Biochim. Biophys. Acta. 2011; 1811: 648-656Crossref PubMed Scopus (169) Google Scholar, 23.Quehenberger O. Armando A.M. Brown A.H. Milne S.B. Myers D.S. Merrill A.H. Bandyopadhyay S. Jones K.N. Kelly S. Shaner R.L. et al.Lipidomics reveals a remarkable diversity of lipids in human plasma.J. Lipid Res. 2010; 51: 3299-3305Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar). Briefly, kidney tissue (∼10 mg of tissue) was homogenized in 1 ml of PBS containing 10% methanol. For the analysis of total (esterified and free) FAs, kidney homogenates (50 μl) were spiked with deuterium-labeled FAs, acidified with 200 μl of 0.1 N hydrochloric acid, and extracted with 500 μl of methanol. Phase separation was achieved by the addition of 500 μl of dichloromethane (CH2Cl2), and the organic phase was removed. The extraction with dichloromethane was repeated, and the combined extracts were dried under argon gas. For saponification, the dried lipids were resuspended in 250 μl of methanol and 250 μl of 4 N potassium hydroxide, vortexed for 30 s, and incubated at 37°C for 1 h. The lipid hydrolysates were then neutralized with 260 μl of 4 N HCl and the free FAs were extracted twice with 2 ml of isooctane, and the combined extracts containing the hydrolyzed FAs were dried under argon before analysis with GC-MS. Free FAs were extracted from 50 μl of the kidney tissue homogenates that were supplemented with a set of deuterated FAs that served as internal standards. The extraction was initiated by the addition of 25 μl of 1 N hydrochloric acid and 500 μl of methanol, and a biphasic solution was formed by the addition of 1 ml of isooctane. The isooctane phase containing the free FA fraction is removed, the extraction is repeated once more, and the combined extracts are dried under argon before analysis with the GC-MS. In preparation for GC-MS analysis, the FAs (both total FA after saponification and free FA extracted from samples and the quantitative standards) were taken up in 25 μl of 1% diisopropylethylamine in acetonitrile and derivatized with 25 μl of 1% pentafluorobenzyl bromide. The FA esters were analyzed on an Agilent 6890N gas chromatograph equipped with an Agilent 5973 mass selective detector (Agilent, Santa Clara, CA) operated in the negative chemical ionization mode. Fatty acid quantitation was achieved by the stable isotope dilution method. Eicosanoids were analyzed by LC-MS, as described before (23.Quehenberger O. Armando A.M. Brown A.H. Milne S.B. Myers D.S. Merrill A.H. Bandyopadhyay S. Jones K.N. Kelly S. Shaner R.L. et al.Lipidomics reveals a remarkable diversity of lipids in human plasma.J. Lipid Res. 2010; 51: 3299-3305Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar, 24.Dumlao D.S. Buczynski M.W. Norris P.C. Harkewicz R. Dennis E.A. High-throughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines.Biochim. Biophys. Acta. 2011; 1811: 724-736Crossref PubMed Scopus (116) Google Scholar). For tissue analysis, eicosanoids were isolated from 0.9 ml of kidney homogenate supplemented with a set of internal standards consisting of 26 deuterated eicosanoids (Cayman Chemical) by solid phase extraction (Strata-X; Phenomenex, Torrance, CA) and eluted into 1 ml of methanol. For eicosanoid analysis in urine, 600 μl of sample were processed identically. The extracted samples were brought to dryness, reconstituted in 100 μL of LC buffer consisting of 63% water, 37% acetonitrile, and 0.02% formic acid, and a 40 μL aliquot was separated by reverse phase LC using a Synergy C18 column (2.1 × 250 mm, 4 μm; Phenomenex). Eicosanoids were analyzed using a tandem quadrupole mass spectrometer (MDS SCIEX 4000 Q Trap; Applied Biosystems, Foster City, CA) via scheduled multiple reaction monitoring in the negative ionization mode. Eicosanoids were identified in samples by matching the multiple reaction monitoring signal and LC retention times with those of pure standards. Data analysis was performed using MultiQuant 2.1 software (Applied Biosystems). Quantitative eicosanoid determination was performed using the stable isotope dilution method. Kidney tissue homogenates were electrophoretically separated on NuPAGE bis-Tris gels (Life Technologies) and transferred onto nitrocellulose membrane (Life Technologies). Antibodies to ELOLV5, cytosolic phospho-PLA2 and secretory PLA2 (Abcam), FADS1/FADS2 (Biorbyt, UK), and actin (Sigma) were used on the blots. Detection was performed with ECL Plus detection reagents (GE Healthcare) after treatment with appropriate secondary antibodies. Results are presented as mean values ± SD. The kidney values were normalized with tissue weight and plasma values were normalized to volume. Urinary metabolites are all normalized to urine creatinine values. All metabolites were log transformed and auto-scaled before undergoing statistical analysis using the MetaboAnalyst version 3.0 software (25.Xia J. Wishart D.S. Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis.Curr. Protoc. Bioinformatics. 2002; 55: 14.10.1-14.10.91Crossref Scopus (1070) Google Scholar). Figures were generated using GraphPad Prism software version 4.03 . The difference between data groups was evaluated for significance using two-way ANOVA and Fisher's least significant difference method post hoc tests for multiple comparisons. A P-value less than 0.05 was defined as statistically significant in the non-MS experiments. To account for multiple comparisons with a large number of lipid metabolites, a false discovery rate (FDR; q value) of <0.05 was considered statistically significant. The highly correlated metabolites derived from a single enzyme pathway from a specific parent FA in each tissue compartment and then were separately aggregated into one secondary variable representative of the corresponding pathway using principal component (PC) analysis. We have then used the PC explaining the highest variance in the variable for further analysis to detect trends using SPSS version 25 (IBM Corp.) (26.Afshinnia F. Zeng L. Byun J. Wernisch S. Deo R. Chen J. Hamm L. Miller E.R. Rhee E.P. Fischer M.J. et al.Elevated lipoxygenase and cytochrome P450 products predict progression of chronic kidney disease.Nephrol. Dial. Transplant. July 25, 2018; (Epub ahead of print.)doi:10.1093/ndt/gfy232Google Scholar). Both cytosolic and secretory PLA2 act on membrane phospholipids to release PUFAs, the precursors of various eicosanoids. Levels of cytosolic phospho-PLA2 (group IV) and secretory PLA2 were both increased with HFD in the kidney, and AICAR treatment decreased their levels (Fig. 1). Targeted lipidomic analysis using MS of kidney tissue revealed unique patterns in the total and free ω3 and ω6 PUFA series that are demonstrated in Fig. 2. The total FA (esterified and unesterified FA) and free FA (unesterified) profiles generated from the kidney of mice fed a low-fat standard chow (STD), HFD, or HFD with the AMPK activator AICAR (HFD+AICAR) are displayed in supplemental Table S1. The total EPA and free EPA are decreased in the kidneys with the HFD (Fig. 2A, B; P < 0.05) and further decreased with AICAR, while both total DHA and free DHA (an n-3 FA similar to EPA) are increased with HFD and reversed with the use of AICAR (Fig. 2C, D; P < 0.05). While DHA and EPA levels are modulated with HFD, the parent PUFA, ALA (total and unesterified), is unchanged. The free and total n-6 FA, DGLA, is decreased in the kidney with HFD, but unchanged with AICAR (Fig. 2E, F). Total AA and free AA in the kidney are increased by HFD, and the free AA decreased with AICAR (Fig. 2G, H). Free and total levels of LA, the precursor for both DGLA and AA, are unchanged by the diet and AICAR in the kidney. Delta-6 desaturase and delta-5 desaturase catalyze desaturations at specific positions of FA substrates. Elongases extend the FA carbon chains by two carbons. The levels of both desaturases were not different between the three groups (supplemental Fig. S1). The eicosanoid metabolites of AA, DHA, and EPA are regulated with HFD in the kidney (Table 1), and specific metabolites are modulated with AICAR (Fig. 3). HFD seems to create a clear increase in COX products of AA in the kidney and a decrease in COX products of EPA. 12-Hydroxyl-hexadecatrienoic acid, which is a COX/P450 product, is increased with HFD and decreased with AICAR (Fig. 3A). The P450 products of DHA, 16,17-epoxy-docosapentaenoic acid (EpDPE) and its product 16-hydroxy-docosahexaenoic acid (HDoHE) and 19,20-EpDPE and its product 19,20-dihydroxy-docosapentaenoic acid (DiHDPA), are increased with HFD, but only the end products 19,20-DiHDPA and 16-HDoHE are decreased with AICAR (Fig. 3B, C). The LOX products of DHA (HDoHEs), 4-HDoHE, 7-HDoHE, 11-HDoHE, and 17-HDoHE and its product, 15(t)-protectin D1 (PD1), are increased with HFD (Fig. 3D–F). NE products of DHA, 8-HDoHE, 10-HDoHE, 13-HDoHE, and 20-HDoHE, are increased with HFD and decreased with AICAR (Fig. 3G, 3H).TABLE 1.Eicosanoid metabolites altered with HFD in the kidneyNameAcronymMECHSTDHFDPq ValueUp with HFDAA metabolitesArachidonic acidAAPUFA243.4 ± 19.2312.7 ± 40.90.0100.03713,14-Dihydro-15-keto-prostaglandin D2dhk-PGD2COX0.04 ± 0.011.20 ± 0.020.0080.0355,6-Epoxyeicosatrienoic acid5,6-EETP4501.59 ± 0.748.64 ± 5.640.0140.04511,12-Epoxyeicosatrienoic acid11,12-EETP4500.02 ± 0.010.18 ± 0.130.0150.0455-Oxo-eicosatetraenoic acid5-oxoETELOX0.22 ± 0.111.56 ± 1.210.0130.0436(R),15(R)-lipoxin A46(R),15(R)-LXA4LOX0.02 ± 0.010.05 ± 0.020.0060.0326(S)-lipoxin A46SLXA4LOX0.02 ± 0.010.05 ± 0.020.0150.0459-Hydroxy-eicosatetraenoic acid9-HETENE0.02 ± 0.010.14 ± 0.090.0130.043DHA metabolites16,17-Epoxy-docosapentaenoic acid16,17-EpDPEP4500.42 ± 0.070.65 ± 0.080.0010.01016-Hydroxy-docosahexaenoic acid16-HDoHEP4500.60 ± 0.071.20 ± 0.24<0.0010.00319,20-Epoxy-docosapentaenoic acid19,20-EpDPEP4501.03 ± 0.191.63 ± 0.220.0010.01019,20-Dihydroxy-docosapentaenoic acid19,20-DiHDPAP4500.55 ± 0.101.32 ± 0.430.0010.0084-Hydroxy-docosahexaenoic acid4-HDoHELOX1.22 ± 0.203.17 ± 1.360.0070.0327-Hydroxy-docosahexaenoic acid7-HDoHELOX0.17 ± 0.020.32 ± 0.090.0040.02111-Hydroxy-docosahexaenoic acid11-HDoHELOX0.25 ± 0.030.45 ± 0.06<0.0010.00117-Hydroxy-docosahexaenoic acid17-HDoHELOX1.04 ± 0.131.84 ± 0.31<0.0010.005Protectin DXPDXLOX0.02 ± 0.010.07 ± 0.020.0090.03715(t)-Protectin D115(t)-PD1LOX4.14 ± 0.8912.68 ± 3.760.0010.0088-Hydroxy-docosahexaenoic acid8-HDoHENE0.67 ± 0.081.16 ± 0.240.0010.00910-Hydroxy-docosahexaenoic acid10-HDoHENE0.26 ± 0.030.45 ± 0.070.0010.00713-Hydroxy-docosahexaenoic acid13-HDoHENE0.51 ± 0.100.78 ± 0.110.0030.01720-Hydroxy-docosahexaenoic acid20-HDoHENE1.61 ± 0.222.46 ± 0.400.0020.013EPA metabolites11-Hydroxy-eicosapentaenoic acid11-HEPENE0.28 ± 0.102.0 ± 1.500.0100.037Down with HFDAA metabolitesProstaglandin E2PGE2COX2.88 ± 1.021.43 ± 0.210.0110.040Dihomo-γ-LA metabolites15-Keto-prostaglandin F1α15-keto-PGF1αCOX0.43 ± 0.090.23 ± 0.080.0070.03215-Hydroxy-eicosatrienoic acid15-HETrELOX0.46 ± 0.070.34 ± 0.040.0090.037EPA metabolites5-Hydroxy-eicosapentaenoic acid5-HEPELOX0.24 ± 0.030.09 ± 0.02<0.0010.00112-Hydroxy-eicosapentaenoic acid12-HEPELOX4.23 ± 0.311.17 ± 0.26<0.001<0.00115-Hydroxy-eicosapentaenoic acid15-HEPELOX0.15 ± 0.020.05 ± 0.01<0.001<0.00118-Hydroxy-eicosapentaenoic acid18-HEPENE0.22 ± 0.030.07 ± 0.01<0.001<0.001All values are picomoles per milligram of renal tissue expressed as mean ± SD. MECH, mechanism. Open table in a new tab All values are picomoles per milligram of renal tissue expressed as mean ± SD. MECH, mechanism. HFD-induced changes in urine metabolites are illustrated in Table 2 and the metabolites that are altered with AICAR in Fig. 4. The overall trend of the kidney continues in the urine with a predominant increase of COX products of AA in the urine and a decrease of COX products of EPA. Adrenic acid and its COX metabolite, dihomo-prostaglandin (PG)F2α (dihomo-PGF2α), are both increased with HFD in the urine, but only dihomo-PGF2α is decreased with AICAR (Fig. 4A). Dihomo-15-deoxy-PGD2 (dihomo-15d-PGD2), which is a NE byproduct of another adrenic acid COX metabolite, is decreased with HFD and increased with AICAR (Fig. 4B).TABLE 2.Eicosanoid metabolites altered with HFD in the urineNameAcronymMECHSTDHFDPq ValueUp with HFDAdrenic acid metabolitesAdrenic acidAdrenic acidPUFA0.018 ± 0.0090.27 ± 0.270.0010.011Dihomo-prostaglandin F2αdihomo-PGF2αCOX0.009 ± 0.0030.62 ± 0.18<0.001<0.001AA metabolitesArachidonic acidAAPUFA1.768 ± 0.61914.7 ± 8.9<0.0010.003Prostaglandin A2PGA2COX0.018 ± 0.0180.09 ± 0.030.0010.0011Prostaglandin B2PGB2COX0.035 ± 0.0090.09 ± 0.09<0.0010.00513,14-Dihydro-15-keto-prostaglandin E2PGEMCOX0.354 ± 0.0351.23 ± 0.17<0.001<0.001Prostaglandin J2PGJ2COX0.002 ± 0.0020.009 ± 0.0040.0020.01713,14-Dihydro-15-keto-prostaglandin D2dhk-PGD2COX0.008 ± 0.0030.026 ± 0.004<0.001<0.0016,15-Diketo-13,14-dihydro-prostaglandin F1α6,15 dk-,dh-PGF1αCOX0.27 ± 0.092.12 ± 0.79<0.0010.001Prostaglandin F2αPGF2αCOX0.001 ± 0.0010.09 ± 0.0260.0040.02313,14-Dihydro-15- keto-prostaglandin F2αPGFMCOX1.59 ± 0.267.25 ± 1.76<0.001<0.0016-Keto-prostaglandin E16kPGE1COX0.09 ± 0.0090.09 ± 0.0170.0020.0138,9-Dihydroxy-eicosatrienoic acid8,9-diHETrE
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