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• W2045971486 abstract "Advanced glycation end-products (AGE) are generated by chronic hyperglycaemia and may cause diabetic microvascular complications such as diabetic nephropathy. Many factors influence the development of diabetic nephropathy; however, dysregulation of mesangial cell (MC) proliferation appears to play an early and crucial role. In this study, we investigated the effects of AGE on rat MC proliferation and the involvement of sphingolipids in the AGE response. Results show a bimodal effect of AGE on MC proliferation. Thus, low AGE concentrations (<1 μm) induced a significant increase (+26%) of MC proliferation, whereas higher concentrations (10 μm) markedly reduced it (–24%). In parallel, AGE exerted biphasic effects on neutral ceramidase expression and activity. Low AGE concentrations increased neutral ceramidase activity and expression, whereas high AGE concentrations showed opposite effects. Surprisingly, neutral ceramidase modulation did not result in changes of ceramide levels. However, the AGE (10 μm)-inhibitory effect on MC proliferation was associated with accumulation of sphingosine and was specifically prevented by blocking glucosylceramide synthesis, suggesting that the high AGE concentration effects are mediated by sphingosine and/or glycolipids. On the other hand, treatment of cells with low AGE concentrations led to an increase of sphingosine kinase activity and sphingosine-1-phosphate production that drove the increase of MC proliferation. Interestingly, in glomeruli isolated from streptozotocin-diabetic rats, a time-dependent modulation of ceramidase activity was observed as compared with controls. These results suggest that AGE regulate MC growth by modulating neutral ceramidase and endogenous sphingolipids. Advanced glycation end-products (AGE) are generated by chronic hyperglycaemia and may cause diabetic microvascular complications such as diabetic nephropathy. Many factors influence the development of diabetic nephropathy; however, dysregulation of mesangial cell (MC) proliferation appears to play an early and crucial role. In this study, we investigated the effects of AGE on rat MC proliferation and the involvement of sphingolipids in the AGE response. Results show a bimodal effect of AGE on MC proliferation. Thus, low AGE concentrations (<1 μm) induced a significant increase (+26%) of MC proliferation, whereas higher concentrations (10 μm) markedly reduced it (–24%). In parallel, AGE exerted biphasic effects on neutral ceramidase expression and activity. Low AGE concentrations increased neutral ceramidase activity and expression, whereas high AGE concentrations showed opposite effects. Surprisingly, neutral ceramidase modulation did not result in changes of ceramide levels. However, the AGE (10 μm)-inhibitory effect on MC proliferation was associated with accumulation of sphingosine and was specifically prevented by blocking glucosylceramide synthesis, suggesting that the high AGE concentration effects are mediated by sphingosine and/or glycolipids. On the other hand, treatment of cells with low AGE concentrations led to an increase of sphingosine kinase activity and sphingosine-1-phosphate production that drove the increase of MC proliferation. Interestingly, in glomeruli isolated from streptozotocin-diabetic rats, a time-dependent modulation of ceramidase activity was observed as compared with controls. These results suggest that AGE regulate MC growth by modulating neutral ceramidase and endogenous sphingolipids. Chronic hyperglycaemia promotes advanced glycation end-products (AGE) 1The abbreviations used are: AGE, advanced glycation end-product(s); BSA, bovine serum albumin; CDase, ceramidase; N-CDase, neutral CDase; Cer, ceramide; DAG, diacylglycerol; DMS, N,N-dimethylsphingosine; MC, mesangial cell(s); NBD-C12-Cer, N-[12-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-d-erythro-sphingosine; OPA, o-phthaldehyde; PBS, phosphate-buffered saline; PPMP, dl-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol; PTX, pertussis toxin; RAGE, receptor for advanced glycation end-products; S1P, sphingosine 1-phosphate; Sph, sphingosine; SphK, sphingosine kinase; STZ, streptozotocin; HPLC, high pressure liquid chromatography; siRNA, small interfering RNA. 1The abbreviations used are: AGE, advanced glycation end-product(s); BSA, bovine serum albumin; CDase, ceramidase; N-CDase, neutral CDase; Cer, ceramide; DAG, diacylglycerol; DMS, N,N-dimethylsphingosine; MC, mesangial cell(s); NBD-C12-Cer, N-[12-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-d-erythro-sphingosine; OPA, o-phthaldehyde; PBS, phosphate-buffered saline; PPMP, dl-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol; PTX, pertussis toxin; RAGE, receptor for advanced glycation end-products; S1P, sphingosine 1-phosphate; Sph, sphingosine; SphK, sphingosine kinase; STZ, streptozotocin; HPLC, high pressure liquid chromatography; siRNA, small interfering RNA. formation and accumulation in vivo. It has been largely documented that glycation, as a nonenzymatic reaction between glucose and amino acid residues of long living proteins, leads to structural and functional alterations of structural as well as circulating proteins, such as hemoglobin, IgG, or albumin (1Kennedy L. Mehl T.D. Riley W.J. Merimee T.J. Diabetologia. 1981; 21: 94-98Crossref PubMed Scopus (91) Google Scholar, 2Danze P.M. Tarjoman A. Rousseaux J. Fossati P. Dautrevaux M. Clin. Chim. Acta. 1987; 166: 143-153Crossref PubMed Scopus (41) Google Scholar, 3Brownlee M. Diabetes Care. 1992; 15: 1835-1843Crossref PubMed Scopus (452) Google Scholar). AGE have been shown to contribute to peripheral microvascular alterations, leading to one of the major complications of diabetes mellitus, diabetic nephropathy and end stage renal failure. Indeed, a number of AGE such as carboxymethyllysine or pentosidine have been identified in kidneys of diabetic patients, and their renal accumulation was positively correlated with the disease's severity (4Sugiyama S. Miyata T. Horie K. Iida Y. Tsuyuki M. Tanaka H. Maeda K. Nephrol. Dial. Transplant. 1996; 11: 91-94Crossref PubMed Scopus (37) Google Scholar). AGE glomerular accumulation was also reported in animal models such as the streptozotocin-induced type I diabetes mellitus in rat (5Bendayan M. Kidney Int. 1998; 54: 438-447Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 6Ling X. Nagai R. Sakashita N. Takeya M. Horiuchi S. Takahashi K. Lab. Invest. 2001; 81: 845-861Crossref PubMed Scopus (48) Google Scholar).Compelling evidence suggests that high ambient glucose-induced AGE accumulation contributes importantly to mesangium pathogenesis (7Forbes J.M. Cooper M.E. Oldfield M.D. Thomas M.C. J. Am. Soc. Nephrol. 2003; 14: 254-258Crossref PubMed Google Scholar). For example, one of the dominant histological features of the diseased or diabetic kidney is the expansion of the extracellular matrix. In this regard, an imbalance in the control of mesangial cell (MC) proliferation appears to play an early and crucial role in the initiation and progression of glomerulosclerosis. After entering an early and limited step of hyperplasia, MC arrest in the G1 phase and undergo de novo synthesis and accumulation of constitutive proteins (such as collagen IV, fibronectin, and laminin B1), leading to cellular hypertrophy (8Wolf G. Kidney Int. 2000; 77: 59-66Abstract Full Text Full Text PDF Scopus (127) Google Scholar). At this point, AGE have been recently shown to inhibit human MC proliferation (9Yamagishi S.I. Inagaki Y. Okamoto T. Amano S. Koga K. Takeuchi M. Makita Z. J. Biol. Chem. 2002; 277: 20309-20315Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar) and to promote apoptosis. Additionally, excessive matrix protein secretion and deposition into the conjunctive space by MC has been associated with autocrine overexpression of transforming growth factor β (10Kim Y.S. Kim B.C. Song C.Y. Hong H.K. Moon K.C. Lee H.S. J. Lab. Clin. Med. 2001; 138: 59-68Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Interestingly, transforming growth factor β overproduction was suggested to be triggered by AGE (11Chen S. Cohen M.P. Lautenslager G.T. Shearman C.W. Ziyadeh F.N. Kidney Int. 2001; 59: 673-681Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar).Sphingolipid metabolism, in particular the ceramide (Cer) pathway, has been proposed to regulate many biological responses, including cell survival and cell death. Cer has also been implicated in the pathogenesis of diabetes (12Summers S.A. Garza L.A. Zhou H. Birnbaum M.J. Mol. Cell. Biol. 1988; 18: 5457-5464Crossref Scopus (360) Google Scholar, 13Shimabukuro M. Higa M. Zhou Y.T. Wang M.Y. Newgard C.B. Unger R.H. J. Biol. Chem. 1998; 273: 32487-32490Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). A balance between Cer and other bioactive metabolites such as sphingosine (Sph) and sphingosine-1-phosphate (S1P) has been pointed out as a sensitive rheostat controlling growth and death of various cell types (14Spiegel S. Merrill Jr., A.H. FASEB J. 1996; 10: 1388-1397Crossref PubMed Scopus (640) Google Scholar, 15Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar). Thus, in contrast to the growth-inhibitory and proapoptotic effects of Cer and Sph (16Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1487) Google Scholar, 17Mathias S. Pena L.A. Kolesnick R.N. Biochem. J. 1998; 335: 465-480Crossref PubMed Scopus (617) Google Scholar, 18Cuvillier O. Biochim. Biophys. Acta. 2002; 1585: 153-162Crossref PubMed Scopus (281) Google Scholar, 19Sweeney E.A. Sakakura C. Shirahama T. Masamune A. Ohta H. Hakomori S. Igarashi Y. Int. J. Cancer. 1996; 66: 358-366Crossref PubMed Scopus (175) Google Scholar, 20Huwiler A. Pfeilschifter J. van den Bosch H. J. Biol. Chem. 1999; 274: 7190-7195Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), S1P has been shown to promote cell growth in various cell types including MC (21Gennero I. Fauvel J. Nieto M. Cariven C. Gaits F. Briand-Mesange F. Chap H. Salles J.P. J. Biol. Chem. 2002; 277: 12724-12734Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 22Katsuma S. Hada Y. Ueda T. Shiojima S. Hirasawa A. Tanoue A. Takagaki K. Ohgi T. Yano J. Tsujimoto G. Genes Cells. 2002; 7: 1217-1230Crossref PubMed Scopus (67) Google Scholar). Many studies have reported the involvement of sphingomyelinases, the ceramide-generating enzymes, in the regulation of Cer levels in response to proinflammatory cytokines, growth factors, or stress stimuli (23Huwiler A. Kolter T. Pfeilschifter J. Sandhoff K. Biochim. Biophys. Acta. 2000; 1485: 63-99Crossref PubMed Scopus (376) Google Scholar, 24Levade T. Jaffrezou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar). Ceramidases (CDases), the ceramide-degrading enzymes, could also play an important role in the regulation of Cer levels. CDases hydrolyze the Cer N-acyl linkage between the fatty acyl and the sphingosine base. Current knowledge suggests that this catabolic pathway represents the prevailing source of cellular Sph (25Merrill Jr., A.H. Wang E. Methods Enzymol. 1992; 209: 427-437Crossref PubMed Scopus (63) Google Scholar, 26Rother J. van Echten G. Schwarzmann G. Sandhoff K. Biochem. Biophys. Res. Commun. 1992; 189: 14-20Crossref PubMed Scopus (130) Google Scholar, 27Michel C. van Echten-Deckert G. Rother J. Sandhoff K. Wang E. Merrill Jr., A.H. J. Biol. Chem. 1997; 272: 22432-22437Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), which can in turn be phosphorylated by sphingosine kinase (SphK) to form S1P. In this way, CDase activity may be a key step in determining the intracellular levels of Cer and Sph/S1P, thus playing a crucial role in the regulation of cell survival or death in response to external stimuli (reviewed in Ref. 28El Bawab S. Mao C. Obeid L.M. Hannun Y.A. Subcell. Biochem. 2002; 36: 187-205Crossref PubMed Google Scholar).Indeed, recent studies have implicated CDase activity in MC proliferation in response to platelet-derived growth factor stimulation (29Coroneos E. Martinez M. McKenna S. Kester M. J. Biol. Chem. 1995; 270: 23305-23309Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Franzen et al. (30Franzen R. Pautz A. Brautigam L. Geisslinger G. Pfeilschifter J. Huwiler A. J. Biol. Chem. 2001; 276: 35382-35389Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) showed that chronic interleukin-1β treatment of rat mesangial cells results in neutral CDase activation, thereby counteracting sphingomyelinase activation, Cer accumulation, and apoptosis. Nikolova-Karakashian et al. (31Nikolova-Karakashian M. Morgan E.T. Alexander C. Liotta D.C. Merrill Jr., A.H. J. Biol. Chem. 1997; 272: 18718-18724Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) reported in primary cultures of rat hepatocytes a dose-dependent effect of interleukin-1β on CDase activity. Further, this CDase regulation provided a “switch” determining the net levels of Cer and Cer-downstream active metabolites, Sph or S1P, and the subsequent effects on expression of specific genes.In the present study, the involvement of sphingolipid metabolism in the growth response of cultured MC exposed to AGE was investigated. Results show that AGE, most likely through the receptor for AGE (RAGE), exerted a bimodal effect on MC proliferation. These effects were associated with modulation of neutral CDase (N-CDase) and SphK activities and resulted in accumulation of S1P at low AGE concentrations and Sph at high concentrations. Interestingly, ex vivo, glomeruli isolated from diabetic animals demonstrated also a time-dependent modulation of CDase activity and expression as compared with controls. To our knowledge, these results are the first to report a bimodal effect of AGE on MC proliferation and to show the involvement of sphingolipids in these responses.EXPERIMENTAL PROCEDURESMaterials—Culture flasks and plates were from BD Biosciences. Fetal calf serum was from Invitrogen. Media and adjuvants used for cell culture were from Sigma. BSA (fragment V, low endotoxin, fatty acid-free), methylglyoxal, N,N-dimethylsphingosine (DMS), dl-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), pertussis toxin (PTX), and streptozotocin (STZ) were from Sigma. All other chemicals and Escherichia coli DAG kinase were from Calbiochem. Lipids (C18- and C17-d-erythro-sphingosine, sphingosine 1-phosphate, ceramide, and N-[12-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-d-erythro-sphingosine (NBD-C12-Cer)) were purchased from Avanti Polar Lipids (Coger, Paris, France). The BCA protein assay kit was from Pierce. The electrophoresis system (Mini-PROTEAN 3 System), polyacrylamide precast gels, and prestained molecular weight standards were from Bio-Rad (Marnes-la-Coquette, France). Polyvinylidene difluoride membranes (pore size 0.45 μm; Immobilon™-P) were from Millipore (Molsheim, France).All solvents, glass-precoated striped TLC plates (20 × 20 cm; silica gel 60), and the Licrospher® 100 RP-18 column (Hibar® prepacked column, RT 250-4) were from Merck KGaA (Darmstadt, Germany). Sterile filtration was performed with acrodisc filters (low protein-binding proteins; 0.2 μm) from Gelman Laboratory (Ann Arbor, MI). PD-10 gel filtration columns (G-25 Sephadex) were from Amersham Biosciences (Orsay, France). l-[14C(U)]serine (165 mCi/mmol), [γ-33P]adenosine trisphosphate (3000 Ci/mmol), and [methyl-3H]thymidine (6.7 Ci/mmol) were from PerkinElmer Life Sciences. Liquid scintillation Pico Fluor-15™ was from Packard Biosciences (Groningen, The Netherlands). HPLC analysis was performed on an 1100 modular three-dimensional liquid chromatography system (Agilent Technologies, Massy, France). Autoradiography and fluorescence acquisition were performed using Storm™ Imaging Systems, ImageMaster™ VDS-CL, and ImageQuant™ software for data quantitative analysis (Amersham Biosciences).Culture of Rat Mesangial Cells—Renal mesangial cells were obtained from young Wistar rat (Charles River, L'Arbresle, France) kidneys. Primary cultures were established from freshly isolated glomeruli cortex fragments, which were mechanically sieved and harvested by iterative selection on specific mesh sizes (230, 73.7, and final 70 μm; Cellector®). Cells were then seeded on fibronectin-coated (20 μg/ml) dishes and cultured under standard conditions at 37 °C in a humidified 5% CO2 incubator in Dulbecco's modified Eagle's medium, 5 mm d-glucose, supplemented with 15% (v/v) heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine. MC were characterized by their morphological appearance in phase contrast and by positive staining for vimentin, α-actin, and Thy-1 antigen, as previously described (32MacKay K. Striker L.J. Elliot S. Pinkert C.A. Brinster R.L. Striker G.E. Kidney Int. 1988; 33: 677-684Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 33Mene P. J. Nephrol. 2001; 14: 198-203PubMed Google Scholar).In order to avoid contamination by residual epithelial or endothelial cells, experiments were performed between the 5th and 12th passage. In all experiments, MC were grown to near confluence in the presence of 15% fetal calf serum and then made quiescent by serum starvation in Dulbecco's modified Eagle's medium with 0.5% fetal calf serum for 24 h. Treatments and analysis were performed on quiescent cells.AGE-BSA Preparation—AGE and control BSA were prepared as described by Westwood et al. (34Westwood M.E. McLellan A.C. Thornalley P.J. J. Biol. Chem. 1994; 269: 32293-32298Abstract Full Text PDF PubMed Google Scholar) and Denis et al. (35Denis U. Lecomte M. Paget C. Ruggiero D. Wiernsperger N. Lagarde M. Free Radic. Biol. Med. 2002; 33: 236-247Crossref PubMed Scopus (119) Google Scholar) by incubating BSA (7.2 mg/ml) with or without methylglyoxal (100 mm) at 37 °C for 50 h under sterile conditions. In order to remove salts and unreacted carbonyls, AGE and control BSA were passed on PD-10 gel filtration columns, and then they were sterile-filtered and stored at –20 °C until use.Proliferation Assay: Thymidine Uptake—A[3H]thymidine incorporation assay was performed to measure the effect of AGE and other compounds on DNA synthesis of MC. Quiescent confluent MC cultured in 6-well plates (about 105 cells/well) were treated with AGE for 72 h and incubated for the last 18 h of treatment with [3H]thymidine (5 μCi/ml). Culture medium was then removed, cells washed twice with ice-cold PBS and briefly washed twice in 5% (w/v) trichloroacetic acid. The acid-insoluble material was dissolved in lysis buffer (0.1 m NaOH, 2% (w/v) Na2CO3, 1% (w/v) SDS), and the incorporated [3H]thymidine was measured by liquid scintillation.Transient Transfection of MC with RAGE siRNA—Blocking RAGE expression was performed using rat RAGE-specific siRNA (Ambion, Huntingdon, UK) and Oligofectamine™ reagent (Invitrogen, Cergy Pontoise, France) to transfect cells. The RAGE antisense sequence of 21 bases used was UCCAAGCUUCAGUCCUUCCtt. MC grown to 40% confluence in 6-well plates were transfected with various concentrations of RAGE siRNA according to the manufacturer's instructions. When cells were treated with AGE-BSA or BSA control (72 h), cells were transfected with 400 nm RAGE siRNA for 24 h before treatment.Enzyme Assays—In these experiments, quiescent confluent MC cultured in 10-cm diameter Petri dishes (about 1.5 × 106 cells/plate) were treated for 12–72 h with AGE or control BSA. Culture medium was then removed, plates were washed twice with ice-cold PBS, and cells were scraped with a rubber policeman in the specified buffer.Ceramidase Assay—Assay was adapted from El Bawab et al. (36El Bawab S. Bielawska A. Hannun Y.A.J. J. Biol. Chem. 1999; 274: 27948-27955Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Briefly, cells were scrapped in 300 μl of cold lysis buffer (25 mm Hepes, pH 7.5, 5 mm EDTA, 0.2% Triton X-100, 1.5 mm sodium fluoride, 1 mm sodium vanadate, 10 μl/ml protease inhibitors mixture (Protease Arrest™; Calbiochem)) and centrifuged for 5 min at 10,000 × g at 4 °C. The supernatant was collected, and protein concentration was determined using the BCA assay. Synthetic fluorescent substrate specific for CDases (NBD-C12-Cer) was solubilized by sonication (250 μm in 0.1% Triton X-100). Each sample (400 μg of proteins) was assayed for N-CDase activity by incubation with 25 μm NBD-C12-Cer in 0.2 m Hepes (pH 7.5) for 2 h at 37 °C. At the end of the incubation, lipids, including released NBD-dodecanoic acid, were extracted with chloroform/methanol (1:1, v/v), spotted onto a TLC plate together with a NBD-dodecanoic acid standard (10–250 pmol), and developed in chloroform/methanol/ammonia (25:5:0.3, v/v/v). NBD-dodecanoic acid was visualized and quantified by fluorescent imaging. CDase-specific activity was expressed in pmol of released fatty acid/min/mg of protein.Sphingosine Kinase Assay—SphK activity was measured as described by Olivera et al. (37Olivera A. Barlow K.D. Spiegel S. Methods Enzymol. 2000; 311: 215-223Crossref PubMed Scopus (70) Google Scholar) with slight modifications. Cells collected in 300 μl of lysis/reaction buffer (20 mm Tris, pH 7.4, 20% glycerol, 1 mm β-mercaptoethanol, 1 mm EDTA, 1 mm sodium vanadate, 15 mm sodium fluoride, 40 mm glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, 0.5 mm 4-deoxypyridoxine, 10 μl/ml protease inhibitor mixture) were frozen-thawed five times and centrifuged for 5 min at 10,000 × g at 4 °C. The supernatant was collected and used for the SphK assay. SphK activity was measured in the presence of 50 μm sphingosine (dissolved in 5% Triton X-100, final concentration 0.25%) and [γ-33P]ATP (10 μCi; 1 mm) containing MgCl2 (10 mm) for 1 h at 37 °C. Reactions were terminated by the addition 10 μl of 1 n HCl and 400 μl of chloroform/methanol/HCl (100:200:1, v/v/v). After vortexing, 120 μl of chloroform and 120 μl of 2 m KCl were added, and organic phase was separated by centrifugation. Dried lipids were spotted on a TLC plate and developed in 1-butanol/ethanol/acetic acid/water (80:20:10:20, v/v/v/v). Plates were then exposed to a Molecular Dynamics PhosphorImager® screen (Amersham Biosciences), and the radioactive spots corresponding to S1P were visualized by autoradiography. Bands corresponding to [33P]S1P were quantified, and SphK specific activity was expressed in arbitrary units/min/mg of protein.Lipid Analysis and Quantification—Quiescent confluent MC cultured in 10-cm Petri dishes were treated for 72 h with AGE or control BSA. Culture medium was removed, cells were washed twice with ice-cold PBS and then scraped in 1 ml of methanol, and total lipids were extracted by the method of Bligh and Dyer (38Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42136) Google Scholar). Total lipids in the organic phase were then dried, and an aliquot was taken from each sample for total lipid phosphate determination as described (39Bielawska A. Perry D.K. Hannun Y.A. Anal. Biochem. 2001; 298: 141-150Crossref PubMed Scopus (76) Google Scholar).Ceramide Measurement Using the Diacylglycerol Kinase Assay— Measurement of Cer levels was performed using the DAG kinase assay as described (39Bielawska A. Perry D.K. Hannun Y.A. Anal. Biochem. 2001; 298: 141-150Crossref PubMed Scopus (76) Google Scholar, 40Perry D.K. Bielawska A. Hannun Y.A. Methods Enzymol. 2000; 312: 22-31Crossref PubMed Google Scholar). Briefly, lipid samples and standards were sonicated in 20 μl of mixed micelles (7.5% β-n-octyl-d-glucopyranoside, 25 mm dioleyl phosphatidylglycerol) and incubated for 30 min at 37 °C. Then 70 μl of enzyme reaction buffer (75 mm imidazole, pH 6.6, 71 mm LiCl, 17.8 mm MgCl2, 1.5 mm EGTA, 0.25 mm diethylenetriaminepentaacetic acid, 2.8 mm dithiothreitol, and 3 μg of Escherichia coli DAG kinase) and 10 μl of ATP mixture (10 mm ATP and 0.2 μCi/μl [γ-33P]ATP in 5 mm imidazole) were added to samples, and the mixture was incubated for 1 h at room temperature. Phosphorylated Cer and DAG were extracted with chloroform/methanol. Lipids and DAG and Cer standards (0–600 pmol) were then spotted onto TLC plates and developed in chloroform/acetone/methanol/acetic acid/water (50:20:15: 10:5, v/v/v/v/v). Radioactive DAG 1-phosphate and Cer 1-phosphate were visualized and quantified by autoradiography.HPLC Assay of S1P and Sph—HPLC assay was used to quantitate the released S1P and Sph, as described by Min et al. (41Min J.M. Yoo H.S. Lee E.Y. Lee W.J. Lee Y.M. Anal. Biochem. 2002; 303: 167-175Crossref PubMed Scopus (139) Google Scholar). Cells from 3 × 10-cm diameter dishes were scraped, pooled, and centrifuged, and pellets were resuspended into 100 μl of PBS, and an aliquot was kept for protein determination. After adding 250 μl of methanol, 0.6 μl of concentrated HCl, and 20 pmol of C17-sphingosine 1-phosphate as an internal standard, samples were sonicated for 5 min at 4 °C. 500 μl of chloroform plus 1 m NaCl (1:1, v/v) and 25 μl of 3 n NaOH were added, and the mixture was vortexed and centrifuged to allow phase separation. S1P is water-soluble at alkaline pH and partitions into the aqueous phase. After centrifugation, the lower phase was re-extracted twice with 250 μl of methanol plus 1 m NaCl (1:1, v/v), 13 μl of 3 n NaOH. Aqueous phases were combined, mixed thoroughly with 130 μl of reaction buffer (200 mm Tris-HCl, pH 7.4, 75 mm MgCl2, 2 m glycine, pH 9), 50 units of alkaline phosphatase and incubated for 1 h at 37 °C on a 200-μl chloroform layer placed at the bottom of the reaction mixture. Dephosphorylated sphingoid bases were extracted twice with 300 μl of chloroform. Pooled organic layers were further washed three times with 300 μl of alkaline water, placed in amber glass microvials, dried under N2, and dissolved in 120 μl of ethanol. Released Sph was derivatized with 15 μl of freshly prepared o-phthaldehyde reagent (OPA) (5 ml of 3% (w/v) boric acid, pH 10.5, 10 μl of β-mercaptoethanol, and 100 μl of ethanol containing 5 mg of OPA). The mixture was allowed to stand for 20 min at room temperature before analysis. HPLC was conducted using a Hewlett Packard 1100-HPLC model fitted with a 5-μm RP-C18 column (25 × 4) combined with a guard column cartridge. The solvent was acetonitrile/water (90:10, v/v), and elution was achieved during 50 min with a flow rate of 1 ml/min. A spectrofluorometer detector was used with an excitation at 340 nm and emission at 455 nm. The retention times of C17- and C18-S1P were around 9 and 12 min, respectively. Chromatographic profiles were then analyzed using the HP Chemstation software. S1P released in the culture medium of MC was measured as described above, except for the volume of solvent used for extraction.For Sph measurement, standard C17-Sph was added in the residual organic phase after primary S1P extraction. Sph derivatization and analysis was then performed as described for S1P.Western Blot Analysis—Quiescent confluent MC in 10-cm diameter dishes were treated with the indicated concentrations of AGE or control BSA for 72 h. The culture medium was removed, and the cells were washed with ice-cold PBS. Cells were then scraped directly into lysis buffer (25 mm Hepes, pH 7.5, 5 mm EDTA, 0.2% Triton X-100, 1.5 mm sodium fluoride, 1 mm sodium vanadate, 10 μl/ml protease inhibitor mixture) and homogenized by brief sonication. The homogenate was centrifuged for 10 min at 10,000 × g, and the supernatant was taken for protein determination. Equal amounts of cell lysates (50–100 μg of protein) were boiled in Laemmli's sample buffer, separated on 7.5% SDS-PAGE, and blotted onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk in 0.1% Tween 20/PBS (PBST) for 1 h, and incubated overnight with an affinity-purified polyclonal rabbit anti-CDase antibody (1:1000). The antibody was raised against the 311–327 peptide KNRGYLPGQGPFVANFA and was a generous gift from Dr. Yusuf A. Hannun (Medical University of South Carolina, Charleston, SC). The next day, membranes were washed for 30 min in 0.1% PBST and incubated in the same buffer containing 5% nonfat dry milk with goat anti-rabbit horseradish peroxidase-conjugated IgG (1:1000) (DAKO, Trappes, France). Immune complexes were detected by enhanced chemiluminescence (ECL™ Western blotting detection reagents kit; Amersham Biosciences). CDase bands were quantified using ImageQuant software, and data are expressed as intensity arbitrary units. Results were corrected for total proteins loaded using α-actin detected separately for each sample lane with a mouse monoclonal antibody (Oncogene Research Products, San Diego, CA) at a dilution of 1:1000.Western blot was also performed to analyze RAGE expression using a primary goat RAGE polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a dilution of 1:1000.Induction of Diabetes in Rats—Wistar male rats, weighing 176/200 g, were injected intravenously with 65 mg/kg STZ in citrate buffer, pH 4.5. Control rats were injected with citrate buffer alone. Animals were housed in cages with standard chow and water ad libitum. STZ and control rats were sacrificed either 4 days or 4 weeks after injection. Blood glucose levels were measured by a glucose-oxidase colorimetric assay, body weight was checked at this point, and the kidneys were harvested. Glomeruli were isolated by mechanical sieving and frozen at –70 °C until further analysis. Glomeruli were resuspended in lysis buffer (25 mm Hepes, pH 7.5, 5 mm EDTA, 0.2% Triton X-100, 1.5 mm sodium fluoride, 1 mm sodium vanadate, protease inhibitor mixture) and homogenized by serial passes through a 23-gauge needle fitted on a 1-ml syringe. After centrifugation at 10,000 × g for 5 min, the supernatant was used for prot" @default.
• W2045971486 created "2016-06-24" @default.
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• W2045971486 title "Bimodal Effect of Advanced Glycation End Products on Mesangial Cell Proliferation Is Mediated by Neutral Ceramidase Regulation and Endogenous Sphingolipids" @default.
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