Lipid Droplet Accumulation Is Associated with an Increase in Hyperglycemia-Induced Renal Damage
2013; Elsevier BV; Volume: 182; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2012.11.033
ISSN1525-2191
AutoresÉva Kiss, Bettina Kränzlin, Katja Wagenblaβ, Mahnaz Bonrouhi, Joachim Thiery, Elisabeth Gröne, Viola Nordström, Daniel Teupser, Norbert Gretz, Ernst Malle, Hermann‐Josef Gröne,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoDyslipidemia is a frequent component of the metabolic disorder of diabetic patients contributing to organ damage. Herein, in low-density lipoprotein receptor–deficient hyperlipidemic and streptozotozin-induced diabetic mice, hyperglycemia and hyperlipidemia acted reciprocally, accentuating renal injury and altering renal function. In hyperglycemic-hyperlipidemic kidneys, the accumulation of Tip47-positive lipid droplets in glomeruli, tubular epithelia, and macrophages was accompanied by the concomitant presence of the oxidative stress markers xanthine oxidoreductase and nitrotyrosine, findings that could also be evidenced in renal biopsy samples of diabetic patients. As liver X receptors (LXRα,β) regulate genes linked to lipid and carbohydrate homeostasis and inhibit inflammatory gene expression in macrophages, the effects of systemic and macrophage-specific LXR activation were analyzed on renal damage in hyperlipidemic-hyperglycemic mice. LXR stimulation by GW3965 up-regulated genes involved in cholesterol efflux and down-regulated proinflammatory/profibrotic cytokines, inhibiting the pathomorphology of diabetic nephropathy, renal lipid accumulation, and improving renal function. Xanthine oxidoreductase and nitrotyrosine levels were reduced. In macrophages, GW3965 or LXRα overexpression significantly suppressed glycated or acetylated low-density lipoprotein–induced cytokines and reactive oxygen species. Specifically, in mice, transgenic expression of LXRα in macrophages significantly ameliorated hyperlipidemic-hyperglycemic nephropathy. The results demonstrate the presence of lipid droplet–induced oxidative mechanisms and the pathophysiologic role of macrophages in diabetic kidneys and indicate the potent regulatory role of LXRs in preventing renal damage in diabetes. Dyslipidemia is a frequent component of the metabolic disorder of diabetic patients contributing to organ damage. Herein, in low-density lipoprotein receptor–deficient hyperlipidemic and streptozotozin-induced diabetic mice, hyperglycemia and hyperlipidemia acted reciprocally, accentuating renal injury and altering renal function. In hyperglycemic-hyperlipidemic kidneys, the accumulation of Tip47-positive lipid droplets in glomeruli, tubular epithelia, and macrophages was accompanied by the concomitant presence of the oxidative stress markers xanthine oxidoreductase and nitrotyrosine, findings that could also be evidenced in renal biopsy samples of diabetic patients. As liver X receptors (LXRα,β) regulate genes linked to lipid and carbohydrate homeostasis and inhibit inflammatory gene expression in macrophages, the effects of systemic and macrophage-specific LXR activation were analyzed on renal damage in hyperlipidemic-hyperglycemic mice. LXR stimulation by GW3965 up-regulated genes involved in cholesterol efflux and down-regulated proinflammatory/profibrotic cytokines, inhibiting the pathomorphology of diabetic nephropathy, renal lipid accumulation, and improving renal function. Xanthine oxidoreductase and nitrotyrosine levels were reduced. In macrophages, GW3965 or LXRα overexpression significantly suppressed glycated or acetylated low-density lipoprotein–induced cytokines and reactive oxygen species. Specifically, in mice, transgenic expression of LXRα in macrophages significantly ameliorated hyperlipidemic-hyperglycemic nephropathy. The results demonstrate the presence of lipid droplet–induced oxidative mechanisms and the pathophysiologic role of macrophages in diabetic kidneys and indicate the potent regulatory role of LXRs in preventing renal damage in diabetes. Altered lipoprotein metabolism and intracellular accumulation of unsaturated free fatty acids, cholesteryl esters, and advanced lipoxidation/glyoxylation end products can accelerate the development and progression of glomerular and tubulointerstitial injury in patients with diabetes mellitus.1Jandeleit-Dahm K. Cao Z. Cox A.J. Kelly D.J. Gilbert R.E. Cooper M.E. Role of hyperlipidemia in progressive renal disease: focus on diabetic nephropathy.Kidney Int Suppl. 1999; 71: S31-S36Abstract Full Text Full Text PDF PubMed Google Scholar, 2Abrass C.K. Lipid metabolism and renal disease.Contrib Nephrol. 2006; 151: 106-121Crossref PubMed Scopus (22) Google Scholar Advanced lipoxidation/glyoxylation end products have been shown to induce expression of chemotactic factors [eg, monocyte chemoattractant protein-1 (MCP-1)] and adhesion molecules (eg, intercellular adhesion molecule 1) on endothelial, mesangial, and tubular epithelial cells, with consequent migration of monocytes/macrophages into the kidney.3Kamanna V.S. Pai R. Roh D.D. Kirschenbaum M.A. Oxidative modification of low-density lipoprotein enhances the murine mesangial cell cytokines associated with monocyte migration, differentiation, and proliferation.Lab Invest. 1996; 74: 1067-1079PubMed Google Scholar, 4Gröne H.J. Walli A.K. Gröne E.F. The role of oxidatively modified lipoproteins in lipid nephropathy.Contrib Nephrol. 1997; 120: 160-175Crossref PubMed Google Scholar, 5Kamanna V.S. Pai R. Ha H. Kirschenbaum M.A. Roh D.D. Oxidized low-density lipoprotein stimulates monocyte adhesion to glomerular endothelial cells.Kidney Int. 1999; 55: 2192-2202Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Growing evidence indicates that macrophages accumulate in diabetic kidneys and contribute to the substantial inflammation and fibrosis that can be observed in diabetic nephropathy.6Tesch G.H. Macrophages and diabetic nephropathy.Semin Nephrol. 2010; 30: 290-301Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar Macrophages are cells at the intersection between lipid metabolism and inflammation.7Shibata N. Glass C.K. Macrophages, oxysterols and atherosclerosis.Circ J. 2010; 74: 2045-2051Crossref PubMed Scopus (83) Google Scholar Excess lipids, through several signaling pathways (eg, NF-κB), can activate macrophages, turn them into foam cells, and increase their production of inflammatory mediators.8Schmid H. Boucherot A. Yasuda Y. Henger A. Brunner B. Eichinger F. Nitsche A. Kiss E. Bleich M. Gröne H.J. Nelson P.J. Schlöndorff D. Cohen C.D. Kretzler M. Modular activation of nuclear factor-κB transcriptional programs in human diabetic nephropathy.Diabetes. 2006; 55: 2993-3003Crossref PubMed Scopus (317) Google Scholar, 9Prieur X. Roszer T. Ricote M. Lipotoxicity in macrophages: evidence from diseases associated with the metabolic syndrome.Biochim Biophys Acta. 2010; 1801: 327-337Crossref PubMed Scopus (74) Google Scholar Specifically, data indicate intracellular lipid droplets as functionally active organelles with roles in cell signaling, regulation of lipid metabolism, and synthesis and secretion of inflammatory mediators10Bozza P.T. Bandeira-Melo C. Mechanisms of leukocyte lipid body formation and function in inflammation.Mem Inst Oswaldo Cruz. 2005; 1: 113-120Crossref Scopus (35) Google Scholar; reactive oxygen species (ROS), cytokines/chemokines (eg, IL-1β and MCP-1), and growth factors (eg, platelet-derived growth factor and transforming growth factor β1) then will induce profibrotic responses leading to organ loss.11Zoja C. Garcia P.B. Remuzzi G. The role of chemokines in progressive renal disease.Front Biosci. 2009; 14: 1815-1822Crossref PubMed Scopus (39) Google Scholar, 12Yonemoto S. Machiguchi T. Nomura K. Minakata T. Nanno M. Yoshida H. Correlations of tissue macrophages and cytoskeletal protein expression with renal fibrosis in patients with diabetes mellitus.Clin Exp Nephrol. 2006; 10: 186-192Crossref PubMed Scopus (34) Google Scholar, 13Vallon V. Thomson S.C. Renal function in diabetic disease models: the tubular system in the pathophysiology of the diabetic kidney.Annu Rev Physiol. 2012; 74: 351-375Crossref PubMed Scopus (224) Google Scholar Liver X receptors (LXRs) α and β are ligand-activated nuclear receptors. Both isoforms are expressed at high levels in macrophages.14Repa J.J. Mangelsdorf D.J. The liver X receptor gene team: potential new players in atherosclerosis.Nat Med. 2002; 8: 1243-1248Crossref PubMed Scopus (332) Google Scholar The renal expression of LXRs was found to be reduced in animal models of type 1 diabetes compared with healthy animals.15Proctor G. Jiang T. Iwahashi M. Wang Z. Li J. Levi M. Regulation of renal fatty acid and cholesterol metabolism, inflammation, and fibrosis in akita and OVE26 mice with type 1 diabetes.Diabetes. 2006; 55: 2502-2509Crossref PubMed Scopus (213) Google Scholar After ligand binding, LXRs form heterodimers with retinoid X receptors and regulate transcription of genes involved in lipid/glucose metabolism and inflammation. Natural ligands of LXRs are derived from the oxidative metabolism of cholesterol (oxysterols), potentially implying that disorders of lipid metabolism influence the transcriptional activity of LXRs and may modulate inflammation.14Repa J.J. Mangelsdorf D.J. The liver X receptor gene team: potential new players in atherosclerosis.Nat Med. 2002; 8: 1243-1248Crossref PubMed Scopus (332) Google Scholar, 16Hong C. Tontonoz P. Coordination of inflammation and metabolism by PPAR and LXR nuclear receptors.Curr Opin Genet Dev. 2008; 18: 461-467Crossref PubMed Scopus (175) Google Scholar Previous studies have shown that LXRs control cholesterol efflux in macrophages inhibiting foam cell formation17Larrede S. Quinn C.M. Jessup W. Frisdal E. Olivier M. Hsieh V. Kim M.J. Van Eck M. Couvert P. Carrie A. Giral P. Chapman M.J. Guerin M. Le Goff W. Stimulation of cholesterol efflux by LXR agonists in cholesterol-loaded human macrophages is ABCA1-dependent but ABCG1-independent.Arterioscler Thromb Vasc Biol. 2009; 29: 1930-1936Crossref PubMed Scopus (153) Google Scholar and reduce lipopolysaccharide-induced expression of inflammatory genes (eg, iNOS, COX2, IL-6). Furthermore, LXRs can prevent the development of atherosclerosis in different rodent models.18Ogawa S. Lozach J. Benner C. Pascual G. Tangirala R.K. Westin S. Hoffmann A. Subramaniam S. David M. Rosenfeld M.G. Glass C.K. Molecular determinants of crosstalk between nuclear receptors and toll-like receptors.Cell. 2005; 122: 707-721Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 19Terasaka N. Hiroshima A. Koieyama T. Ubukata N. Morikawa Y. Nakai D. Inaba T. T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice.FEBS Lett. 2003; 536: 6-11Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar Recently, we reported pronounced anti-inflammatory and antifibrotic effects of LXR activation in chronic renal allograft dysfunction and pointed to the substantial contribution of LXR-modulated inflammatory activity of macrophages in achieving these effects.20Kiss E. Popovic Z. Bedke J. Wang S. Bonrouhi M. Gretz N. Stettner P. Teupser D. Thiery J. Porubsky S. Adams J. Gröne H.J. Suppression of chronic damage in renal allografts by liver X receptor (LXR) activation: relevant contribution of macrophage LXRα.Am J Pathol. 2011; 179: 92-103Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar We sought to evaluate the effects of LXR activation by a specific synthetic agonist, GW3965, on the development of diabetic nephropathy and to dissect the contribution of macrophage LXRα. A mouse model of hyperlipidemia-aggravated hyperglycemia was taken as an animal model to mimic a metabolic state regularly present in patients with diabetes mellitus. To gain insight into the role of macrophages, mice specifically expressing LXRα in macrophages were studied. The concomitant presence of the oxidative stress markers xanthine oxidoreductase (XOR) and nitrotyrosine with tail-interacting protein of 47 kDa (Tip47)-positive lipid droplets could be shown in glomerular cells, tubular epithelia, and macrophages in kidneys of these hyperlipidemic-hyperglycemic mice and in renal biopsy samples of diabetic patients. These data, in congruence with the increased production of ROS and inflammatory/fibrotic mediators by lipid-loaded macrophages and tubular epithelial cells, provide insight into the mechanisms of intracellular lipid accumulation–mediated renal lesions, which can be effectively regulated by LXRs. The results demonstrate that LXR activation can prevent the development of hyperlipidemia/hyperglycemia-induced renal lesions by coordinated modulation of lipid metabolism and inflammation. The remarkable potency of LXRα in macrophages to protect kidneys from diabetic injury confirms the importance of the macrophage population in this renal disease and marks LXRs as in vivo relevant modulators and therapeutic targets of macrophage functions in diabetic nephropathy. LDLR−/− mice (C57BL/6 background; The Jackson Laboratory, Bar Harbor, ME) were crossed with mice with transgenic expression of LXRα in macrophages (C57BL/6 background, termed mLXRα-tg)21Teupser D. Kretzschmar D. Tennert C. Burkhardt R. Wilfert W. Fengler D. Naumann R. Sippel A.E. Thiery J. Effect of macrophage overexpression of murine liver X receptor-α (LXR-α) on atherosclerosis in LDL-receptor deficient mice.Arterioscler Thromb Vasc Biol. 2008; 28: 2009-2015Crossref PubMed Scopus (59) Google Scholar to generate LDLR−/− mLXRα-tg animals. Mice were initially maintained on a pelleted rodent chow diet, and at 7 weeks of age they were randomly assigned to one of the four diets: chow diet, Western diet (WD), chow + streptozotocin (STZ), and WD + STZ. The experimental animal groups were as follows: LDLR−/−, LDLR−/− with GW3965, and LDLR−/− mLXRα-tg (Table 1). In addition, mLXRα-tg mice and C57BL/6 wild-type (WT) mice were evaluated as control animals and are listed in Supplemental Table S1.Table 1Biochemical Data in the Experimental GroupsGroupBW (g)Kidney/BW ratio (%)Plasma glucose (mg/dL)Plasma cholesterol (mg/dL)Plasma triglyceride (mg/dL)1. LDLR−/− 1a. Chow (n = 6)30.8 ± 1.50.59 ± 0.01196.7 ± 8.2456.5 ± 52.2192.7 ± 15.7 1b. WD (n = 6)39.1 ± 1.9∗P < 0.05. vs 1a0.62 ± 0.06229.8 ± 17.31075.0 ± 128.8†P < 0.01. vs 1a277.3 ± 92.5 1c. Chow + STZ (n = 8)27.1 ± 0.6∗P < 0.05. vs 1a,†P < 0.01. vs 1b0.68 ± 0.02†P < 0.01. vs 1a410.7 ± 55.9†P < 0.01. vs 1a, 1b602.2 ± 59.9205.0 ± 20.3 1d. WD + STZ (n = 6)24.1 ± 0.9∗P < 0.05. vs 1a, 1b0.83 ± 0.10 vs 1a403.6 ± 55.6†P < 0.01. vs 1a, 1b1514.0 ± 182.7†P < 0.01. vs 1a, 1c377.0 ± 103.7∗P < 0.05. vs 1a2. LDLR−/− + GW3965 2a. Chow (n = 4)31.4 ± 0.60.60 ± 0.01234.8 ± 11.5438.5 ± 21.7468.3 ± 45.6†P < 0.01. vs 1a 2b. WD (n = 6)31.6 ± 1.3∗P < 0.05. vs 1b0.57 ± 0.02233.7 ± 15.9971.2 ± 268.6∗P < 0.05. vs 2a279.4 ± 75.8 2c. Chow + STZ (n = 5)24.9 ± 1.2∗P < 0.05. vs 2a,†P < 0.01. vs 2b0.69 ± 0.03492.0 ± 71.7∗P < 0.05. vs 2a, 2b445.5 ± 52.1346.8 ± 90.4 2d. WD + STZ (n = 6)25.5 ± 1.2†P < 0.01. vs 2a,∗P < 0.05. vs 2b0.59 ± 0.03403.0 ± 64.9∗P < 0.05. vs 2a, 2b1205.0 ± 275.0†P < 0.01. vs 2a, 2c361.7 ± 52.13. LDLR−/− mLXRa-tg 3a. Chow (n = 6)29.4 ± 0.90.58 ± 0.04212.3 ± 6.9296.7 ± 22.6∗P < 0.05. vs 1a,†P < 0.01. vs 2a130.2 ± 8.8†P < 0.01. vs 1a, 2a 3b. WD (n = 6)34.2 ± 1.3∗P < 0.05. vs 3a0.63 ± 0.03207.3 ± 18.5395.8 ± 64.1∗P < 0.05. vs 1b133.8 ± 19.9 3c. Chow + STZ (n = 7)22.2 ± 1.2†P < 0.01. vs 3a, 3b, 1c0.77 ± 0.07585.5 ± 95.7†P < 0.01. vs 3a, 3b426.0 ± 23.4†P < 0.01. vs 3a173.4 ± 29.4 3d. WD + STZ (n = 7)25.4 ± 1.2∗P < 0.05. vs 3a,†P < 0.01. vs 3b0.78 ± 0.04∗P < 0.05. vs 3a617.2 ± 12.6†P < 0.01. vs 3a, 3b, 1d,∗P < 0.05. vs 2d1496.0 ± 235.0†P < 0.01. vs 3a,∗P < 0.05. vs 3b, 3c345.3 ± 86.7Data are given as means ± SEM.BW, body weight.∗ P < 0.05.† P < 0.01. Open table in a new tab Data are given as means ± SEM. BW, body weight. Rodent chow diet contained 4% fat, 24% protein, and 4.5% crude fiber (8604 Teklad rodent diet; Harlan Laboratories, Indianapolis, IN). The WD contained 21% fat and 0.15% cholesterol (TD 88137 Teklad custom research diet; Harlan Laboratories). The synthetic LXR agonist GW3965 (Sigma-Aldrich, Schnelldorf, Germany, and partly synthesized according to the method of Collins et al22Collins J.L. Fivush A.M. Watson M.A. Galardi C.M. Lewis M.C. Moore L.B. Parks D.J. Wilson J.G. Tippin T.K. Binz J.G. Plunket K.D. Morgan D.G. Beaudet E.J. Whitney K.D. Kliewer S.A. Willson T.M. Identification of a nonsteroidal liver X receptor agonist through parallel array synthesis of tertiary amines.J Med Chem. 2002; 45: 1963-1966Crossref PubMed Scopus (361) Google Scholar) was mixed into rodent chow diet or WD, and mice received 20 mg/kg body weight per day. The diets were fed for 20 weeks. Hyperglycemia was induced by 40 mg/kg body weight i.p. injection of the islet toxin STZ for 5 consecutive days at 7 and 9 weeks of age. The non-STZ groups were injected with citrate buffer as controls. All the animals had free access to tap water. Animal experiments were performed according to German laws on animal protection. Mice were sacrificed by cervical dislocation, and whole animals were perfused with PBS (pH 7.4) via the left ventricle. Kidneys were cut into 1-mm coronal slices and were fixed in 4% formaldehyde in PBS or zinc solution for histologic and immunhistologic analyses and in Karnovsky`s solution [2% paraformaldehyde and 2.5% glutaraldehyde in 0.2 mol/L sodium cacodylate buffer (pH 7.2)] for electron microscopy. In addition, tissue slices were snap frozen in liquid nitrogen and were stored at −80°C. Morphometric analysis was performed using a semiautomatic image analyzing system (Leica Q600 Qwin; Leica Microsystems, Cambridge, UK). Mesangial matrix increase was determined on PAS-stained 3-μm kidney slices (50 glomeruli per slice) by the point-counting method. Results were expressed as a fraction of glomerular surface area.23Adams J. Kiss E. Arroyo A.B. Bonrouhi M. Sun Q. Li Z. Gretz N. Schnitger A. Zouboulis C.C. Wiesel M. Wagner J. Nelson P.J. Gröne H.J. 13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Interstitial scarred area was quantified in Masson`s trichrome–stained kidney slices. Ten randomly selected fields (100×) of cortex and outer medulla were evaluated. Results were expressed as a percentage of the total tubulointerstitial area, obtained after exclusion of glomeruli.23Adams J. Kiss E. Arroyo A.B. Bonrouhi M. Sun Q. Li Z. Gretz N. Schnitger A. Zouboulis C.C. Wiesel M. Wagner J. Nelson P.J. Gröne H.J. 13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Foam cells were evaluated in 50 glomeruli according to the following classification: 0 (no foam cells), 0.5 to 1 ( 75% of the glomerular convolute). A final score was calculated as the sum of indices obtained by multiplication of the percentage of glomeruli with a respective degree of injury with the degree of injury (the percentage of glomeruli with 0.5 was multiplied by 0.5, that of degree 1 × 1, that of degree 2 × 2, and that of degree 3 × 3).23Adams J. Kiss E. Arroyo A.B. Bonrouhi M. Sun Q. Li Z. Gretz N. Schnitger A. Zouboulis C.C. Wiesel M. Wagner J. Nelson P.J. Gröne H.J. 13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar To judge the renal accumulation of neutral fat, frozen slices were stained with oil red O. The thickness of the glomerular basement membrane (GBM) was estimated using the orthogonal intercept method.24Hirose K. Osterby R. Nozawa M. Gundersen H.J. Development of glomerular lesions in experimental long-term diabetes in the rat.Kidney Int. 1982; 21: 689-695Abstract Full Text PDF PubMed Scopus (233) Google Scholar, 25Guo M. Ricardo S.D. Deane J.A. Shi M. Cullen-McEwen L. Bertram J.F. A stereological study of the renal glomerular vasculature in the db/db mouse model of diabetic nephropathy.J Anat. 2005; 207: 813-821Crossref PubMed Scopus (70) Google Scholar Shortly, a subsample of the area of glomerular profiles (three glomeruli of a representative kidney slice per group) was photographed in a systematic independent manner, covering approximately 30% of the total profile area. A systematic line grid was superimposed over the electron microscopy micrographs. Where the grid lines transected the endothelial surface of the GBM, measurements were made of the shortest distance between the endothelial plasma membrane and the plasma membrane of the podocyte foot processes, and a harmonic mean GBM thickness was calculated as described.24Hirose K. Osterby R. Nozawa M. Gundersen H.J. Development of glomerular lesions in experimental long-term diabetes in the rat.Kidney Int. 1982; 21: 689-695Abstract Full Text PDF PubMed Scopus (233) Google Scholar, 25Guo M. Ricardo S.D. Deane J.A. Shi M. Cullen-McEwen L. Bertram J.F. A stereological study of the renal glomerular vasculature in the db/db mouse model of diabetic nephropathy.J Anat. 2005; 207: 813-821Crossref PubMed Scopus (70) Google Scholar Immunohistochemical staining was done on sections of paraffin-embedded kidney samples. The following antibodies were used: rat anti-mouse monoclonal antibodies against F4/80 (Serotec, Oxford, UK), Mac-2/galectin-3 (Acris Antibodies GmbH, Hiddenhausen, Germany), CD3 (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit anti-mouse WT1 (C-19; Santa Cruz Biotechnology), and desmin (GeneTex Inc., San Antonio, TX) and mouse ascites fluid against α-smooth muscle actin (α-SMA; Sigma-Aldrich, St Louis, MO). An alkaline phosphatase/anti–alkaline phosphatase detection system was applied for the immune stainings (Dako, Hamburg, Germany). Control experiments were performed by omitting the primary antibody. Positive glomerular cells were counted in ≥50 glomeruli and were given as the mean per glomerular slice; interstitial positive cells were counted in 20 high-power fields (×40) of cortex and outer medulla and were recorded as mean per high-power field. Polyclonal guinea pig antibodies recognizing Tip47, perilipin, and XOR purchased from Progen Biotechnik GmbH (Heidelberg, Germany), and rabbit antibody for nitrotyrosine epitopes (Millipore, Temecula, CA) were used on formalin-fixed mouse kidney slices and human kidney biopsy samples as well as formalin-fixed mouse peritoneal macrophages (4% paraformaldehyde; 5 minutes). The avidin-biotin complex detection method was applied. Staining intensities were evaluated semiquantitatively in whole mouse kidney slices or in 100 randomly chosen macrophages: 0, no staining; 1, mild staining; 2, moderate staining; and 3, strong staining, and a score was calculated as described for the evaluation of foam cells. Peritoneal macrophages were collected with 10 mL of RPMI 1640 medium (Sigma-Aldrich, Taufkirchen, Germany) from male C57BL/6 (WT) and mLXRα-tg mice 72 hours after i.p. injection of 2 mL of thioglycolate (4%). Cells were seeded at 2 × 106 per well. After 12 hours of incubation at 37C° in 5% CO2, the macrophages were thoroughly washed, and adherent cells were used for experiments. Purity was controlled by flow cytometry and Giemsa staining. Generation of murine bone marrow–derived macrophages was performed according to standard protocols.26Weischenfeldt J. Porse B. Bone marrow-derived macrophages (BMM): isolation and applications.CSH Protoc. 2008; http://dx.doi.org/10.1101/pdb.prot5080PubMed Google Scholar, 27Gersuk G.M. Razai L.W. Marr K.A. Methods of in vitro macrophage maturation confer variable inflammatory responses in association with altered expression of cell surface dectin-1.J Immunol Methods. 2008; 329: 157-166Crossref PubMed Scopus (35) Google Scholar In brief, mouse femurs were dissected, and each bone was flushed with 10 mL of PBS. A bone marrow cell suspension sample was collected and centrifuged. Pellets were resuspended in RPMI 1640 medium supplemented by 20% macrophage colony-stimulating factor–containing L929 medium. The cells were plated on non–tissue-culture-treated 10-cm Petri dishes and were incubated at 37°C in 5% CO2. Fresh medium was provided on days 3 and 5, and the experiments were performed on day 7. After preincubation with dimethyl sulfoxide or 3 μmol/L GW3965 or 10 μmol/L 22(R)-OH-cholesterol (Sigma-Aldrich, Taufkirchen, Germany) dissolved in dimethyl sulfoxide for 16 hours, macrophages were stimulated with 30 μg/mL of glycated low-density lipoprotein (gLDL) or 50 μg/mL of acetylated LDL (acLDL) for 12, 24, or 48 hours. The cells were then washed and collected for RNA isolation or were fixed on coverslips for 5 minutes with 4% paraformaldehyde for immunostaining. Mouse peritoneal macrophages seeded onto coverslips and fixed with paraformaldehyde were blocked with 10% fetal calf serum/PBS 0.05% Tween and incubated with primary antibodies: rabbit-α Tip47 and goat-α XOR (both from Santa Cruz Biotechnology) for 1 hour at 37C°. After washing, they were stained with secondary antibodies (Alexa Fluor 488 and 546; Invitrogen, Darmstadt, Germany) (1 hour at 37C°). Cells were also stained with DAPI (Sigma-Aldrich, Taufkirchen, Germany) to visualize the nuclei. Negative controls were performed by omitting the primary antibody. Proximity ligation assay, which allows visualization, localization, and quantification of individual protein interactions at a range of 30 to 40 nm, was performed according to the manufacturer's guidelines (Duolink orange detection system; Olink Bioscience, Uppsala, Sweden).28Söderberg O. Gullberg M. Jarvius M. Ridderstråle K. Leuchowius K.J. Jarvius J. Wester K. Hydbring P. Bahram F. Larsson L.G. Landegren U. Direct observation of individual endogenous protein complexes in situ by proximity ligation.Nat Methods. 2006; 3: 995-1000Crossref PubMed Scopus (1672) Google Scholar Formation of proximity ligation assay spots was analyzed by fluorescence microscopy (Zeiss Cell Observer; Carl Zeiss MicroImaging GmbH, Jena, Germany). Staining intensities were evaluated semiquantitatively in five randomly chosen fields (×200) (280 to 300 cells per group): 0, no staining; 0.5, moderate staining; and 1, strong staining, and a score was calculated as described for the evaluation of foam cells. ROS/reactive nitrogen species were detected using an ROS detection kit (Enzo Life Sciences, Lörrach, Germany) according to the manufacturer's instructions. This assay is designed to directly monitor real-time ROS/reactive nitrogen species production in live cells.29Huo X. Juergens S. Zhang X. Rezaei D. Yu C. Strauch E.D. Wang J.Y. Cheng E. Meyer F. Wang D.H. Zhang Q. Spechler S.J. Souza R.F. Deoxycholic acid causes DNA damage while inducing apoptotic resistance through NF-κB activation in benign Barrett's epithelial cells.Am J Physiol Gastrointest Liver Physiol. 2011; 301: G278-G286Crossref PubMed Scopus (88) Google Scholar Peritoneal and bone marrow–derived macrophages (2 × 104 cells per well) and HK2 immortalized human proximal tubular cells (5 × 103 cells per well; ATCC, Manassas, VA) were seeded in black-walled 96-well plates and were treated as described previously herein. Plates were read after 10 and 60 minutes of stimulation with 50 μg/mL of acLDL using a FLUOstar Optima multiwell plate fluorescent reader (BMG Labtech GmbH, Offenburg, Germany) equipped with a standard green (490/525 nm) and red (490/580 nm) filter (Optima Technologies, Atlanta, GA). Total RNA was extracted from kidneys and cells using the method of Chomczynski and Sacchi30Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62845) Google Scholar (n = 4 to 6 animals per group). RNA quality was checked using an RNA 6000 Nano Chip (Agilent Technologies, Waldbronn, Germany). Ten micrograms of total RNA was digested with DNase I according to the standard protocol. Three micrograms of total RNA (DNA free) was used for the first-strand cDNA synthesis using SuperScript II Reverse Transcriptase and oligo(dT)12-18 as primer (LifeTechnologies, Karlsruhe, Germany). Real-time PCR was performed by LightCycler using LightCyler-FastStart DNA Master SYBR green I kit (Roche Diagnostics, Mannheim, Germany) as described.23Adams J. Kiss E. Arroyo A.B. Bonrouhi M. Sun Q. Li Z. Gretz N. Schnitger A. Zouboulis C.C. Wiesel M. Wagner J. Nelson P.J. Gröne H.J. 13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar The primer sequences for target genes are shown in Table 2.Table 2Sequences of Primers Used for Real-Time RT-PCRGeneSenseAntisenseGAPDH5′-ACTCCCACTCTTCCACCTTC-3′5′-GGTCCAGGGTTTCTTTACTCC-3′S18r5′-TGCCCTATCAACTTTCGATGGTA-3′5′-CAATTACAGGGCCTCCAAAGAGT-3′RAGE5′-CCATCCTACCTTCTCCTG-3′5′-AGCGACTATTCCACCTTC-3′TNF-a5′-GCTTTCCGAATTCACTGGAG-3′5′-TTGCACCTCAGGGAAGAATC-3′MCP-15′-ACCAAGCTCAAGAGAGAGG-3′5′-ACATTCAAAGGTGCTGAAGAC-3′Collagen I5′-GAGCGGAGAGTACTGGATCG-3′5′-GTTCGGGCTGATGTACCAGT-3′ABCA15′-CCAGACAGTTGTGGATGTGG-3′5′-GACCTCGCTCTTCCTTCCTT-3′ABCG15′-CTTGCAGTAGGGGCTTTCAG-3′5′-GCAAGGCTAGAGGTGTGGAG-3′SR-A15′-GCACAGGATGCAGACAGAAA-3′5′-TGGTCCATCTTGGTGACAGA-3′ Open table in a new tab Plasma glucose levels (using retrobulbar venous plexus blood) were monitored every week (after induction of hyperglycemia) using an Accu-Chek system (Roche Diagnostics). Continuous glucose levels higher than 250 mg/dL were considered diabetic. For measurements of renal functional parameters from blood and urine, animals were kept in metabolic cages for 24 hours. Levels of creatinine in serum and urine (enzymatic determination using the test kit Creatinine Plus Version 2 (Roche Diagnostics),31Keppler A. Gretz N. Schmidt R. Kloetzer H.M. Groene H.J. Lelongt B. Meyer M. Sadick M. Pill J. Plasma creatinine determin
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