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• W1996450462 abstract "Diabetic nephropathy (DN) is a major life-threatening complication of diabetes. Renal lesions affect glomeruli and tubules, but the pathogenesis is not completely understood. Phospholipids and glycolipids are molecules that carry out multiple cell functions in health and disease, and their role in DN pathogenesis is unknown. We employed high spatial resolution MALDI imaging MS to determine lipid changes in kidneys of eNOS−/−db/db mice, a robust model of DN. Phospholipid and glycolipid structures, localization patterns, and relative tissue levels were determined in individual renal glomeruli and tubules without disturbing tissue morphology. A significant increase in the levels of specific glomerular and tubular lipid species from four different classes, i.e., gangliosides, sulfoglycosphingolipids, lysophospholipids, and phosphatidylethanolamines, was detected in diabetic kidneys compared with nondiabetic controls. Inhibition of nonenzymatic oxidative and glycoxidative pathways attenuated the increase in lipid levels and ameliorated renal pathology, even though blood glucose levels remained unchanged. Our data demonstrate that the levels of specific phospho- and glycolipids in glomeruli and/or tubules are associated with diabetic renal pathology. We suggest that hyperglycemia-induced DN pathogenic mechanisms require intermediate oxidative steps that involve specific phospholipid and glycolipid species. Diabetic nephropathy (DN) is a major life-threatening complication of diabetes. Renal lesions affect glomeruli and tubules, but the pathogenesis is not completely understood. Phospholipids and glycolipids are molecules that carry out multiple cell functions in health and disease, and their role in DN pathogenesis is unknown. We employed high spatial resolution MALDI imaging MS to determine lipid changes in kidneys of eNOS−/−db/db mice, a robust model of DN. Phospholipid and glycolipid structures, localization patterns, and relative tissue levels were determined in individual renal glomeruli and tubules without disturbing tissue morphology. A significant increase in the levels of specific glomerular and tubular lipid species from four different classes, i.e., gangliosides, sulfoglycosphingolipids, lysophospholipids, and phosphatidylethanolamines, was detected in diabetic kidneys compared with nondiabetic controls. Inhibition of nonenzymatic oxidative and glycoxidative pathways attenuated the increase in lipid levels and ameliorated renal pathology, even though blood glucose levels remained unchanged. Our data demonstrate that the levels of specific phospho- and glycolipids in glomeruli and/or tubules are associated with diabetic renal pathology. We suggest that hyperglycemia-induced DN pathogenic mechanisms require intermediate oxidative steps that involve specific phospholipid and glycolipid species. The global epidemic of diabetes is a major health problem. Diabetic nephropathy (DN) can develop in about 1/3 of diabetic individuals and is characterized by specific glomerular and tubular lesions in the kidney. These lesions are associated with progression to end stage renal disease with subsequent requirement for renal dialysis and transplantation (1Collins A.J. Foley R.N. Chavers B. Gilbertson D. Herzog C. Johansen K. Kasiske B. Kutner N. Liu J. St. Peter W. et al.United States Renal Data System 2011 Annual Data Report: Atlas of chronic kidney disease & end-stage renal disease in the United States.Am. J. Kidney Dis. 2012; 59: e1-e420Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Despite the significance of DN, there is still incomplete understanding of the pathogenic mechanisms, particularly those underlying the differential susceptibility to DN. Lipids may play a role in DN, but to date, the research focus has been on neutral lipids such as triacylglycerols and cholesterol (2Rutledge J.C. Ng K.F. Aung H.H. Wilson D.W. Role of triglyceride-rich lipoproteins in diabetic nephropathy.Nat. Rev. Nephrol. 2010; 6: 361-370Crossref PubMed Scopus (99) Google Scholar). Phospho- and glycolipids are two major classes of lipid molecules that carry out many biological functions ranging from regulation of physical properties of cellular membranes to cell signaling (3Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation.Physiol. Rev. 2013; 93: 1019-1137Crossref PubMed Scopus (977) Google Scholar, 4Kinnunen P.K. Kaarniranta K. Mahalka A.K. Protein-oxidized phospholipid interactions in cellular signaling for cell death: from biophysics to clinical correlations.Biochim. Biophys. Acta. 2012; 1818: 2446-2455Crossref PubMed Scopus (49) Google Scholar). In diabetes, changes in the levels of these lipids in blood and tissues cause dysregulation of different cellular processes associated with pathogenesis (3Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation.Physiol. Rev. 2013; 93: 1019-1137Crossref PubMed Scopus (977) Google Scholar, 5Weijers R.N. Lipid composition of cell membranes and its relevance in type 2 diabetes mellitus.Curr. Diabetes Rev. 2012; 8: 390-400Crossref PubMed Scopus (115) Google Scholar, 6Galadari S. Rahman A. Pallichankandy S. Galadari A. Thayyullathil F. Role of ceramide in diabetes mellitus: evidence and mechanisms.Lipids Health Dis. 2013; 12: 98Crossref PubMed Scopus (128) Google Scholar, 7Russo S.B. Ross J.S. Cowart L.A. Sphingolipids in obesity, type 2 diabetes, and metabolic disease.Handb. Exp. Pharmacol. 2013; 2013: 373-401Crossref Scopus (72) Google Scholar, 8Ramanadham S. Hsu F-F. Zhang S. Bohrer A. Ma Z. Turk J. Electrospray ionization mass spectrometric analysis of INS-1 insulinoma cell phospholipids. Comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion.Biochim. Biophys. Acta. 2000; 1484: 251-266Crossref PubMed Scopus (37) Google Scholar, 9Hsu F.F. Bohrer A. Wohltmann M. Ramanadham S. Ma Z.M. Yarasheski K. Turk J. Electrospray ionization mass spectrometric analyses of changes in tissue phospholipid molecular species during the evolution of hyperlipidemia and hyperglycemia in Zucker diabetic fatty rats.Lipids. 2000; 35: 839-854Crossref PubMed Google Scholar). Thus, phospho- and glycolipids may have a role in DN. Uncovering molecular events that define mechanisms of susceptibility and progression in DN requires knowledge of the identity and spatial localization of biomolecules within glomerular and tubular areas of the kidney. Such knowledge can be obtained using MALDI imaging MS (IMS), a rapidly advancing technology that acquires molecular information from thin tissue sections in a spatially-defined manner (10Caprioli R.M. Farmer T.B. Gile J. Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS.Anal. Chem. 1997; 69: 4751-4760Crossref PubMed Scopus (1685) Google Scholar, 11Chaurand P. Norris J.L. Cornett D.S. Mobley J.A. Caprioli R.M. New developments in profiling and imaging of proteins from tissue sections by MALDI mass spectrometry.J. Proteome Res. 2006; 5: 2889-2900Crossref PubMed Scopus (227) Google Scholar). The levels and spatial localization of biomolecules can be detected from a single tissue section without the need for specific antibodies or a priori knowledge of what molecules are present. For an imaging experiment, a chemical matrix to aid in the absorption of laser energy and ionization is applied uniformly over the sample. The laser is moved in a raster pattern and a spectrum is collected at every pixel in an ordered array across the tissue. Data can then be displayed as molecular maps of the spatial localization of given m/z values throughout the tissue. Molecular identification can be performed directly on the tissue section by MS/MS analysis. The present study is the first report of the application of MALDI IMS to investigate molecular changes in renal glomerular and tubular phospho- and glycolipids in DN. We utilized a set of experimental tools: a robust DN mouse model, which develops renal lesions comparable to those found in human disease (12Zhao H.J. Wang S. Cheng H. Zhang M.Z. Takahashi T. Fogo A.B. Breyer M.D. Harris R.C. Endothelial nitric oxide synthase deficiency produces accelerated nephropathy in diabetic mice.J. Am. Soc. Nephrol. 2006; 17: 2664-2669Crossref PubMed Scopus (288) Google Scholar); a high spatial resolution MALDI IMS technology; and pyridoxamine (PM), which was employed to elucidate whether hyperglycemia-induced oxidative pathways play a role in phospho- and glycolipid changes relevant to DN. PM is an inhibitor of oxidative and glycoxidative reactions and has been shown to act via sequestration of redox active metal ions, scavenging of reactive carbonyl compounds, and scavenging of hydroxyl radical both in vitro and in vivo (13Voziyan P.A. Hudson B.G. Pyridoxamine as a multifunctional pharmaceutical: targeting pathogenic glycation and oxidative damage.Cell. Mol. Life Sci. 2005; 62: 1671-1681Crossref PubMed Scopus (155) Google Scholar, 14Degenhardt T.P. Alderson N.L. Arrington D.D. Beattie R.J. Basgen J.M. Steffes M.W. Thorpe S.R. Baynes J.W. Pyridoxamine inhibits early renal disease and dyslipidemia in the streptozotocin-diabetic rat.Kidney Int. 2002; 61: 939-950Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 15Voziyan P. Brown K.L. Chetyrkin S. Hudson B. Site-specific AGE modifications in the extracellular matrix: a role for glyoxal in protein damage in diabetes.Clin. Chem. Lab. Med. 2014; 52: 39-45Crossref PubMed Scopus (25) Google Scholar, 16Chetyrkin S.V. Mathis M.E. Ham A.J. Hachey D.L. Hudson B.G. Voziyan P.A. Propagation of protein glycation damage involves modification of tryptophan residues via reactive oxygen species: inhibition by pyridoxamine.Free Radic. Biol. Med. 2008; 44: 1276-1285Crossref PubMed Scopus (69) Google Scholar, 17Voziyan P.A. Khalifah R.G. Thibaudeau C. Yildiz A. Jacob J. Serianni A.S. Hudson B.G. Modification of proteins in vitro by physiological levels of glucose: pyridoxamine inhibits conversion of Amadori intermediate to advanced glycation end-products through binding of redox metal ions.J. Biol. Chem. 2003; 278: 46616-46624Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 18Voziyan P.A. Metz T.O. Baynes J.W. Hudson B.G. A post-Amadori inhibitor pyridoxamine also inhibits chemical modification of proteins by scavenging carbonyl intermediates of carbohydrate and lipid degradation.J. Biol. Chem. 2002; 277: 3397-3403Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). We determined molecular changes at the level of a single glomerulus or tubule, which has not been achieved in the previous studies of renal tissues using MALDI IMS (19Marsching C. Eckhardt M. Gröne H-J. Sandhoff R. Hopf C. Imaging of complex sulfatides SM3 and SB1a in mouse kidney using MALDI-TOF/TOF mass spectrometry.Anal. Bioanal. Chem. 2011; 401: 53-64Crossref PubMed Scopus (47) Google Scholar, 20Ruh H. Salonikios T. Fuchser J. Schwartz M. Sticht C. Hochheim C. Wirnitzer B. Gretz N. Hopf C. MALDI imaging MS reveals candidate lipid markers of polycystic kidney disease.J. Lipid Res. 2013; 54: 2785-2794Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 21Kaneko Y. Obata Y. Nishino T. Kakeya H. Miyazaki Y. Hayasaka T. Setou M. Furusu A. Kohno S. Imaging mass spectrometry analysis reveals an altered lipid distribution pattern in the tubular areas of hyper-IgA murine kidneys.Exp. Mol. Pathol. 2011; 91: 614-621Crossref PubMed Scopus (23) Google Scholar). Our data demonstrated that the levels of specific phospho- and glycolipids in glomeruli and/or tubules of the kidney are associated with diabetic renal pathology. Inhibition of glycoxidative pathways, without lowering hyperglycemia, ameliorated lipid levels and renal lesions. We suggest that hyperglycemia-induced DN pathogenic mechanisms require intermediate oxidative steps that involve phospho- and glycolipids. Animal experiments were performed at Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited animal facilities at Vanderbilt University Medical Center according to Institutional Animal Care and Use Committee (IACUC)-approved experimental protocol. Mice were housed in a pathogen-free barrier facility and given standard chow (Lab Diet 5015; PMI Nutrition International, Richmond, IN) and water ad libitum. Upon development of hyperglycemia (about 6 weeks of age), eNOS−/− C57BLKS db/db mice were randomized according to body weight and assigned to either diabetic or diabetic/PM treatment groups. Mice in the diabetic/PM treatment group received PM in drinking water at a daily dose of 400 mg/kg body weight, based on previously published reports of PM protection from kidney injury in diabetic mice (22Zheng F. Zeng Y.J. Plati A.R. Elliot S.J. Berho M. Potier M. Striker L.J. Striker G.E. Combined AGE inhibition and ACEi decreases the progression of established diabetic nephropathy in B6 db/db mice.Kidney Int. 2006; 70: 507-514Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To minimize possible chemical degradation of PM, a light-sensitive compound, fresh solutions were prepared twice a week and administered in water bottles wrapped in aluminum foil as previously described (23Chetyrkin S.V. Kim D. Belmont J.M. Scheinman J.I. Hudson B.G. Voziyan P.A. Pyridoxamine lowers kidney crystals in experimental hyperoxaluria: A potential therapy for primary hyperoxaluria.Kidney Int. 2005; 67: 53-60Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). PM treatment continued until the mice were euthanized at 22 weeks of age. The control group included wild-type C57BLKS mice. Kidneys were removed and either fixed for histological analyses by light and electron microscopy or flash-frozen in liquid nitrogen and stored at −80°C for IMS analyses. Glucose levels were measured in blood collected from the tail vein using OneTouch glucometer and Ultra test strips (LifeScan, Milpitas, CA) as previously described (12Zhao H.J. Wang S. Cheng H. Zhang M.Z. Takahashi T. Fogo A.B. Breyer M.D. Harris R.C. Endothelial nitric oxide synthase deficiency produces accelerated nephropathy in diabetic mice.J. Am. Soc. Nephrol. 2006; 17: 2664-2669Crossref PubMed Scopus (288) Google Scholar, 24Kanetsuna Y. Takahashi K. Nagata M. Gannon M.A. Breyer M.D. Harris R.C. Takahashi T. Deficiency of endothelial nitric-oxide synthase confers susceptibility to diabetic nephropathy in nephropathy-resistant inbred mice.Am. J. Pathol. 2007; 170: 1473-1484Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Albumin and creat­inine excretion was determined in spot urine collected from individually caged mice using Albuwell-M kits (Exocell Inc., Philadelphia, PA) as previously described (12Zhao H.J. Wang S. Cheng H. Zhang M.Z. Takahashi T. Fogo A.B. Breyer M.D. Harris R.C. Endothelial nitric oxide synthase deficiency produces accelerated nephropathy in diabetic mice.J. Am. Soc. Nephrol. 2006; 17: 2664-2669Crossref PubMed Scopus (288) Google Scholar, 24Kanetsuna Y. Takahashi K. Nagata M. Gannon M.A. Breyer M.D. Harris R.C. Takahashi T. Deficiency of endothelial nitric-oxide synthase confers susceptibility to diabetic nephropathy in nephropathy-resistant inbred mice.Am. J. Pathol. 2007; 170: 1473-1484Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The assay variability was <5% in duplicate measurements. Renal histology was assessed in mice at 22 weeks of age. The kidneys were removed and fixed overnight in 10% formalin at 4°C, and 3 μm-thick sections were stained with periodic acid-Schiff (PAS) and Jones' silver staining. Histological evaluation by light microscopy was performed without knowledge of the identity of the various groups. A semi-quantitative index was used to evaluate the degree of glomerular mesangial expansion and sclerosis. Each glomerulus on a single section was graded from 0 to 3, where 0 represents no lesion, and 1, 2, and 3 represent mesangial matrix expansion or sclerosis involving <25, 25 to 50, and >50% of the glomerular tuft area, respectively (see supplementary Fig. II). For electron microscopy, kidneys were cut into small tissue blocks (1 mm3) and fixed in 2.5% glutaraldehyde fixative with 0.1 mol/l cacodylate buffer (pH 7.4) overnight at 4°C. After postfixation with 1% osmium tetroxide, tissues were dehydrated in a series of graded ethanol preparations and embedded in epoxy resin (Poly/Bed 812 embedding media; Polysciences, Warrington, PA). Ultrathin sections were stained with uranyl acetate and lead citrate. Sections were observed by transmission electron microscopy (H-7000; Hitachi, Tokyo, Japan) at 75 kV to determine tubular basement membrane and glomerular basement membrane (GBM) thickness. Immunohistochemical detection of fibronectin was performed using an anti-fibronectin antibody (Sigma, St. Louis, MO). The kidney sections then were incubated using the avidin-biotin-horseradish peroxidase technique (Elite Vectastain ABC kit; Vector Laboratories, Burlingame, CA), and staining was visualized using 3,3′-diaminobenzidine. Frozen kidneys were sectioned on a cryostat at 8 μm thickness, thaw mounted on conductive indium tin oxide-coated glass slides, and dried in a desiccator. The tissue sections were washed by dipping the slide in 50 mM ammonium formate at 4°C three times for five seconds each to remove salts and increase the sensitivity for lipid analysis (25Angel P.M. Spraggins J.M. Baldwin H.S. Caprioli R. Enhanced sensitivity for high spatial resolution lipid analysis by negative ion mode matrix assisted laser desorption ionization imaging mass spectrometry.Anal. Chem. 2012; 84: 1557-1564Crossref PubMed Scopus (161) Google Scholar). MALDI matrix was applied using a custom-built sublimation apparatus which uses reduced pressure and heat for vapor deposition of the MALDI matrix on to the sample slide (26Hankin J.A. Barkley R.M. Murphy R.C. Sublimation as a method of matrix application for mass spectrometric imaging.J. Am. Soc. Mass Spectrom. 2007; 18: 1646-1652Crossref PubMed Scopus (444) Google Scholar) resulting in a uniform MALDI matrix coating over the tissue. 1,5-Diaminonaphthalene was sublimed at 110°C and 50 mTorr for 7 min. The resulting matrix coating contained 0.13 ± 0.02 mg 1,5-diaminonaphthalene/cm2. MALDI imaging experiments were performed in negative ion mode using a Bruker Ultraflextreme TOF mass spectrometer in reflectron geometry. Spectra were collected in the range of m/z 400–1,500 with 10 shots/spectra. Raster steps were taken in 10 μm stage increments and the laser spot size was also 10 μm in diameter as measured on a thin matrix coating. Each imaging experiment was run as a set containing a kidney section from each experimental group. Areas of the cortex were selected for imaging of approximately 20,000 pixels per kidney. Reproducibility of the IMS measurements within a single mouse was assessed by analyzing three separate sections from the same kidney. Lipid measurements by IMS were highly reproducible as shown in supplementary Fig. III. FlexImaging was used for image visualization. Frozen kidneys were sectioned as above. 9-Aminoacridine MALDI matrix was applied by sublimation at 140°C and 50 mTorr for 12 min. The resulting matrix coating contained 1.1 ± 0.2 mg 9-aminoacridine/cm2. MALDI imaging experiments were performed in negative ion mode using a 9.4 T SolariX MALDI Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (Bruker Daltonics). Spectra were collected in the range of m/z of 400–1,500 with 500 shots/spectra. Image resolution was set at 40 μm or 10 μm. FlexImaging and DataAnalysis were used for image visualization and data analysis. After all imaging experiments, the MALDI matrix was removed from the slides by immersion in 70% ethanol followed by 95% ethanol for 30 s each. Kidney sections were then stained with PAS and renal tissue structures were matched to MALDI IMS data via image overlay. All lipids reported were identified using MS/MS fragmentation along with accurate mass data. Accurate masses were determined after imaging experiments by profiling an adjacent tissue section using MALDI FTICR MS. Phospholipid species were isolated and fragmented with the FTICR mass spectrometer using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) for identification. Glycolipid species MS/MS fragmentation experiments were performed on a MALDI-LTQ-XL hybrid linear ion trap instrument (Thermo Scientific) using pulsed q-dissociation. The LIPID MAPS database (lipidmaps.org) was used to search the accurate mass data. Fragmentation patterns were interpreted manually along with tools from lipidmaps.org. For the MALDI imaging datasets, ImageJ software (National Institutes of Health, Bethesda, MD) was used to measure the relative abundance of the lipid species of interest between experimental groups. Monochromatic TIFF images were exported from FlexImaging to ImageJ. Areas of interest were selected in each image as individual glomeruli, tubules, or the entire kidney section. Signal intensity was measured as the mean intensity per area of interest (i.e., glo­meruli, tubules, or entire kidney cross-section). Glomerular and tubular signals were evaluated as single ions and the Amadori-phosphatidylethanolamines (PEs) were evaluated as the ratio of the Amadori-PE signal to the unmodified PE signal. Frozen kidneys were sectioned as above and processed using a washing procedure described by Yang and Caprioli (27Yang J. Caprioli R.M. Matrix sublimation/recrystallization for imaging proteins by mass spectrometry at high spatial resolution.Anal. Chem. 2011; 83: 5728-5734Crossref PubMed Scopus (283) Google Scholar). Histology-directed IMS was performed as described previously (28Cornett D.S. Mobley J.A. Dias E.C. Andersson M. Arteaga C.L. Sanders M.E. Caprioli R.M. A novel histology-directed strategy for MALDI-MS tissue profiling that improves throughput and cellular specificity in human breast cancer.Mol. Cell. Proteomics. 2006; 5: 1975-1983Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), with some modifications. Kidney sections were stained with 0.5% cresyl violet for 30 s followed by an ethanol rinse. Coordinates of the individual glomeruli and tubules within the kidney sections were recorded using a Mirax slide scanner (Zeiss). Trypsin and the MALDI matrix were deposited using an automated acoustic robotic spotter (Portrait 630, Labcyte). Trypsin solution (76 ng/μl trypsin in 100 mM ammonium bicarbonate/10% acetonitrile) was spotted on the tissue in single droplets (∼120 pL) for a total of 40 iterations. Proteolytic digestion was allowed to take place for 2 h. Subsequently, α-cyano-4-hydroxycinnamic acid MALDI matrix (10 mg/ml α-cyano-4-hydroxycinnamic acid in 1:1 v/v mixture of acetonitrile and 0.2% trifluoroacetic acid) was robotically spotted on the trypsin-digested glomeruli. MALDI MS analyses of tryptic peptides were acquired on a Bruker UltrafleXtreme mass spectrometer in positive ion reflectron mode. Analysis was performed with 250 shots/spectra. Digestion peptide profiles from the glomeruli were acquired in the range of m/z 600–3,900. Peaks with significant intensity changes between the groups were manually selected for MS/MS analysis. MS/MS spectra were submitted to the MASCOT (Matrix Science) database search engine to match tryptic peptide sequences to their respective intact proteins (seesupplementary Fig. IV). The intensity of the fibronectin peptide at m/z 1,906 was used to determine the relative deposition of fibronectin in glomeruli from different experimental groups (Fig. 2F). This increase in fibronectin deposition by IMS was consistent with that determined using the classical immunohistochemistry approach (supplementary Fig. V). Data were expressed as mean ± SEM and statistical analysis was performed using Student's t-test for unpaired samples or ANOVA followed by post hoc Student-Newman-Keuls comparisons. For the MALDI imaging datasets, the mean and standard error were calculated for each MS peak of interest. Differences were evaluated by the Kruskal-Wallis rank-sum test followed by post hoc Tukey test. Differences were considered statistically significant at P < 0.05. We employed eNOS−/− C57BLKS db/db mice, the most robust mouse model of type 2 DN to date. At >20 weeks of age, these mice exhibit albuminuria, arteriolar hyalinosis, increased GBM thickness, mesangial expansion, mesangiolysis, focal segmental and early nodular glomerulosclerosis, and markedly decreased glomerular filtration rate (12Zhao H.J. Wang S. Cheng H. Zhang M.Z. Takahashi T. Fogo A.B. Breyer M.D. Harris R.C. Endothelial nitric oxide synthase deficiency produces accelerated nephropathy in diabetic mice.J. Am. Soc. Nephrol. 2006; 17: 2664-2669Crossref PubMed Scopus (288) Google Scholar). In our study, eNOS−/− C57BLKS db/db mice developed significant albuminuria at 6 weeks of age, which increased dramatically by 22 weeks of age (Fig. 1). Treatment of diabetic mice with PM significantly ameliorated albuminuria at 22 weeks of age (Fig. 1). Kidneys of three animals from each treatment group were taken for MALDI IMS analyses of lipids. The second set of kidneys from the same three animals in each treatment group was subjected to renal pathology analyses to allow for direct comparison of renal injury and lipid profiles. Diabetic mice exhibited a dramatic increase in glomerular and tubular pathologic lesions (Fig. 2B–F). PM treatment significantly ameliorated these lesions (Fig. 2B–F). Interestingly, PM treatment did not inhibit hyperglycemia itself (Fig. 2A). Therefore, use of PM treatment allowed us to compare renal lipid profiles in hyperglycemic animals with significantly different degrees of renal pathology. MALDI IMS was performed on renal sections from three biological replicates in each experimental group (nondiabetic, diabetic, and diabetic + PM). Because DN lesions affect primarily glomeruli and tubules, we focused on the lipid molecular patterns localized specifically within glomerular and tubular areas of the renal cortex. We examined 60–70 glomeruli and/or tubules per mouse in each experimental group. Multiple species that belong to different lipid classes were identified within glomerular and tubular structures (supplementary Table I). We then focused only on those specific phospho- and glycolipid species that exhibited significant changes in glomerular and/or tubular levels in diabetes compared with control. These species belonged to four lipid classes: gangliosides, sulfoglycosphingolipids, lysophospholipids, and PEs, and are highlighted in supplementary Table I. Gangliosides are anionic glycosphingolipids located to the outer leaflet of plasma membranes and characterized by the presence of sialic acid in their structure (29van Echten G. Sandhoff K. Ganglioside metabolism. Enzymology, topology, and regulation.J. Biol. Chem. 1993; 268: 5341-5344Abstract Full Text PDF PubMed Google Scholar). Gangliosides are known to play major roles in cell-cell and cell-matrix recognition via interactions with integrins, matrix proteins, and other glycosphingolipids, as well as in innate immunity, apoptosis, and carcinogenesis (30Varki N.M. Varki A. Diversity in cell surface sialic acid presentations: implications for biology and disease.Lab. Invest. 2007; 87: 851-857Crossref PubMed Scopus (403) Google Scholar, 31Schauer R. Sialic acids as regulators of molecular and cellular interactions.Curr. Opin. Struct. Biol. 2009; 19: 507-514Crossref PubMed Scopus (508) Google Scholar, 32Malykh Y.N. Schauer R. Shaw L. N-Glycolylneuraminic acid in human tumours.Biochimie. 2001; 83: 623-634Crossref PubMed Scopus (240) Google Scholar). We determined renal localization and levels of two abundant mammalian gangliosides, N-acetylneuraminic acid (NeuAc)-monosialodihexosylganglioside (GM3) (m/z 1,151.7) and its hydroxylated derivative N-glycolylneuraminic acid (NeuGc)-GM3 (m/z 1,167.7) (Fig. 3). Both ganglioside species were localized exclusively to renal glomeruli (Fig. 3A, B). However, there was a distinct difference in the response of these species to our experimental treatments. NeuAc-GM3 was detected at relatively high levels that were not significantly different in all treatment groups (Fig. 3A, top row; Fig. 3D). In contrast, NeuGc-GM3 was present at relatively low levels in the glomeruli of nondiabetic animals but increased ∼8-fold in the glomeruli of diabetic mice (Fig. 3A, bottom row; Fig. 3D). Diabetic mice treated with PM had significantly lower levels of NeuGc-GM3 compared with untreated diabetic mice (Fig. 3A, bottom row; Fig. 3D). Sulfoglycolipids are produced from glycosphingolipids via addition of one or several sulfate esters catalyzed by the enzyme cerebroside sulfotransferase. Sulfoglycolipids are essential in such key biological processes as nerve fiber myelination and spermatogenesis (33Honke K. Zhang Y. Cheng X. Kotani N. Taniguchi N. Biological roles of sulfoglycolipids and pathophysiology of their deficiency.Glycoconj. J. 2004; 21: 59-62Crossref PubMed Scopus (59) Google Scholar). They are also enriched in mammalian kidneys, where they have been shown to be involved in osmoregulation and acid-base homeostasis (34Niimura Y. Moue T. Takahashi N. Nagai K. Medium osmolarity-dependent biosynthesis of renal cellular sulfoglycolipids is mediated by the MAPK signaling pathway.Biochim. Biophys. Acta. 2010; 1801: 1155-1162Crossref PubMed Scopus (4) Google Scholar, 35Stettner P. Bourgeois S. Marsching C. Traykova-Brauch M. Porubsky S. Nordstrom V. Hopf C. Kosters R. Sandhoff R. Wiegandt H. et al.Sulfatides are required for renal adaptation to chronic metabolic acidosis.Proc. Natl. Acad. Sci. USA. 2013; 110: 9998-10003Crossref PubMed Scopus (46) Google Scholar). We have identified several species of sulfoglycolipids localized specifically to mouse renal tubules" @default.
• W1996450462 created "2016-06-24" @default.
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• W1996450462 date "2014-07-01" @default.
• W1996450462 modified "2023-10-12" @default.
• W1996450462 title "Diabetic nephropathy induces alterations in the glomerular and tubule lipid profiles" @default.
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