In multicellular organisms, cell growth and differentiation is controlled in part by apoptosis. One major apoptotic pathway is triggered by Fas receptor (Fas)-Fas ligand (FasL) interaction. Resistance to Fas-mediated apoptosis is regulated through the production of a soluble Fas isoform (sFas), created by exclusion of transmembrane spanning sequences encoded within exon 6 (FasΔ6) that can bind FasL and block apoptosis. Long noncoding RNAs (lncRNAs) are >200-nucleotide sequences that are important regulators of cellular programs. However, their role in erythropoiesis is just beginning to be appreciated with studies limited to murine systems. I studied the potential role of lncRNAs during human red blood cell development using RNA from cultured CD34+ purified from fetal liver (FL), cord blood (CB), or adult bone marrow (BM) to screen 82 documented lncRNAs. This screen revealed that the Fas Anti-sense (Saf) was consistently increased during maturation and levels high for BM compared to FL or CB. Next, I characterized the regulatory sequence of Saf and by In silico analysis identified canonical binding sites for the erythroid-specific transcription factors KLF1 and GATA1. Chromatin immunoprecipitation (ChIP) assays confirmed binding of both factors to their target sequences and luciferase reporter constructs revealed synergistic activity evidenced by increase in luciferase expression relative to controls. Genome wide expression analysis using cells with overexpression of Saf showed no effect on global gene transcription, suggesting Saf function at post transcriptional levels. Saf was shown to participate in alternative splicing of Fas pre-mRNA through unknown mechanisms. Using a combination of biochemical assays, overexpression and knockdown studies, I showed how this occurs. Cell fractionation and RT-PCR demonstrated that Saf is localized in the nucleus. I found that Saf directly interacted with Fas pre-mRNA to form RNaseA-resistant double-stranded RNA intermediates at regions that flank Exon6. Post-transcriptional function of Saf was confirmed by qRT-PCR demonstrating significantly increased levels of sFas for Saf overexpressing cells. Enrichment for sFas RNA coincided with reduced Fas on the cell surface and increased sFas protein levels when conditioned supernatants were assayed by ELISA. Conversely, siRNA-mediated knockdown of Saf significantly reduced sFas production compared to non-targeting siRNA controls. Saf-interacting proteins were identified by mixing in vitro transcribed and biotin-labeled Saf RNA with nuclear lysates followed by mass spectrometry analysis. This screen identified human splicing factor 45 (SPF45) which has a known role in Fas pre-mRNA alternative splicing. Specific SPF45/Saf interaction was confirmed by RNA pulldown and western blot with SPF45-specific antibodies and the ability to detect sequences for Saf, Fas and sFas by RT-PCR of RNA that immunoprecipitated with SPF45. SPF45 knockdown decreased sFas transcripts and this reduction corresponded to limited production of sFas and increased sensitivity to Fas-mediated apoptosis when cells were exposed to the Fas-activating antibody CH11. Importantly, overexpression of Saf in SPF45 knockdown cells failed to rescue production of sFas supporting the hypothesis that Saf and SPF45 co-participate in modulating Fas pre-mRNA splicing. Protein phosphorylation modulates the interaction of the splicing factors with RNA. The effect of phosphorylation on the Saf-SPF45 interaction was evaluated using stable cell lines expressing a myc-tagged SPF45 protein or versions modified to introduce alanine in place of threonine 71 (T-71-A) or serine 222 (S-222-A) to prevent phosphorylation. Mutation in S-222-A reduced the interaction of SPF45 with Saf. I conclude that Saf interacts with Fas pre-mRNA at sequences that flank exon 6 and recruits phosphorylated SPF45 as mechanism to recognize exon 6 and allow for alternative splicing of Fas Pre-mRNA. Collectively, these studies reveal a novel mechanism to regulate apoptosis that may be responsible for cell proliferation and drug resistance.

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Made available in DSpace on 2014-06-11T19:24:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-31Bitstream added on 2014-06-13T19:52:06Z : No. of bitstreams: 1 cornelio_alg_me_arafo.pdf: 397515 bytes, checksum: b29bef30bd070aadb818ff553343600a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A proposta deste estudo foi investigar a citotoxicidade, ação antimicrobiana e pH do cimento Portland puro (CP) e associações com agentes radiopacificadores: óxido de bismuto (CPBi), óxido de zircônio (CPZir), tungstato de cálcio (CPCa). Para avaliar o potencial citotóxico, foram empregadas linhagens celulares de fibroblastos do ligamento periodontal de camundongos (mPDL) e osteosarcoma de ratos (ROS 17/2.8). Ambas foram expostas por 24 horas a diferentes concentrações do CP fresco, CP associado com radiopacificadores e cimento de óxido de zinco eugenol. Peróxido de hidrogênio foi aplicado como controle positivo para apoptose. A viabilidade após incubação com os cimentos foi avaliada pela atividade da enzima desidrogenase mitocondrial. A morfologia celular foi analisada microscopicamente pelo corante violeta de cresilo, e o mecanismo de morte celular foi determinado pela metodologia de laranja de acridina/brometo de etídio. Os dados foram analisados estatisticamente por ANOVA e Tukey post-test (p<0.01). A correlação entre os dois tipos de morte celular e valores de pH foi estabelecido pela correlação linear de Pearson. O ensaio da enzima desidrogenase mitocondrial revelou um padrão significante de morte celular apenas nas altas concentrações dos eluídos de cimento. CP puro não foi citotóxico, mesmo na alta concentração de 100mg/ml. As imagens microscópicas mostraram que nenhuma das formulações de CP causaram danos as linhagens celulares. Análises estatísticas dos dados de apoptose/necrose demonstram que CP e CP mais agentes radiopacificadores promoveram morte por necrose estatisticamente significativa apenas em 100mg/ml. Os resultados mostraram que CP associado com óxido de bismuto, óxido de zircônio ou tungstato de cálcio não foram citotóxicos para mPDL ou ROS 17/2.8, e podem ser boas... / The purpose of this study was to investigate the cytotoxicity, antimicrobial and pH of pure Portland cement (PC), and associations with radiopacifier agents: bismuth oxide (CPBi), zirconium oxide (CPZir), calcium tungstate (CPCA). To assess the potential cytotoxicity, fibroblast cell lines from the periodontal ligament of mice (mPDL) and rat osteosarcoma (ROS 17/2.8) were used. Both were exposed for 24 hours with different concentrations of fresh PC, PC associated with radiopacifiers and eugenol zinc oxide cement. Hydrogen peroxide was used as a positive control for apoptosis. The viability after incubation with the cements was evaluated by mitochondrial dehydrogenase enzyme activity. Cell morphology was examined microscopically by cresyl violet stain, and the mechanism of cell death was determined by the method of acridine orange / ethidium bromide. The data were statistically analyzed by ANOVA and Tukey post-test (p <0.01). The correlation between the two types of cell death and pH values was established by Pearson linear correlation. The mitochondrial dehydrogenase enzyme assay revealed a significant pattern of cell death only at high concentrations of the eluted cement. Pure PC was not cytotoxic, even at high concentration of 100mg/ml. Microscopic images showed that none of the formulations of PC caused damage cell lines. Statistical analysis of apoptosis/necrosis data showed that PC and PC plus radiopacifiers agents promoted death by necrosis statistically significant only at 100mg/ml. The results showed that PC associated with bismuth oxide, zirconium oxide or calcium tungstate were not toxic to ROS 17/2.8 or mPDL, and may be good alternatives as radiopacifier agents. The antimicrobial and pH of Portland cement and radiopacifier agents were evaluated. For antimicrobial activity agar diffusion was... (Complete abstract click electronic access below)

nÃo hà / Auxemma oncocalyx Taub pertence a famÃlia das Boraginaceae, à conhecida como âpau brancoâ e frequentemente encontrada no estado do CearÃ. A casca da Ãrvore à um adstringente e popularmente utilizado no tratamento de feridas. Oncocalixona A (Onco-A), uma quinona isolada do extrato etÃlico da A. oncocalyx, possui uma sÃrie de propriedades farmacolÃgicas, tais como: analgÃsica, anti-inflamatÃria, antioxidante, citotÃxica e antitumoral. O presente estudo foi realizado para avaliar os efeitos citotÃxicos da Onco-A na linhagem tumoral promielocÃtica, HL-60. O potencial citotÃxico foi avaliado pelo teste colorimÃtrico de anÃlise indireta, MTT, depois de 24 horas de exposiÃÃo das cÃlulas HL-60 Ãs concentraÃÃes crescentes (8, 16,5 e 33 ÂM) da Onco-A. ApÃs o tratamento, os procedimentos experimentais realizados para identificar os mecanismos de aÃÃo in vitro (no citÃmetro de fluxo) foram de viabilidade celular, externalizaÃÃo da fosfatildiserina, fragmentaÃÃo do DNA intercucleossomal, identificaÃÃo da via apoptÃtica, geraÃÃo de espÃcie reativa de oxigÃnio; os experimentos de anÃlise morfolÃgica foram com azul de tripan, May-Grunwald-Giemsa, e laranja de acridina/ brometo de etÃdio; a integridade do DNA pelo teste cometa e tambÃm avaliado a interaÃÃo da Onco-A com enzima topoisomerase. Resultados: de acordo com o teste do MTT, Onco-A apresentou significante citotoxicidade nas linhagem HL-60 (IC50 11 ÂM). As anÃlises dos experimentos demonstraram que as cÃlulas tratadas com a menor concentraÃÃo de Onco-A tiveram reduÃÃo no volume celular, formaÃÃo de corpos apoptÃticos, condensaÃÃo da cromatina, externalizaÃÃo de fosfatildiserina, reduÃÃo da viabilidade celular e fragmentaÃÃo do DNA. Onco-A causou despolarizaÃÃo mitocondrial, ativou caspase iniciadoras -9 e -8, e as efetoras -3 e -7, clivou a proteÃna Poli ADP-Ribose polimerase â via intrÃnseca e extrÃnseca. Apesar de nossos resultados demonstrem que nenhuma das doses foi capaz de gerar espÃcies reativas de oxigÃnio, depois de prÃ-tratadas por 1 hora com N-AcetilcisteÃna, a citotoxicidade da Onco-A foi alterada, apresentando aumento na integridade da membrana, diminuiÃÃo na fragmentaÃÃo do DNA, parada na fase do ciclo celular G2/M e baixo nÃvel de dano no DNA. Embora a Onco-A nÃo interaja com as enzimas topoisomerases, os dados apresentados sugerem que seu alvo, como aceptor de Michael, pode ser a molÃcula de DNA. Esses resultados sugerem que a apoptose à uma importante forma de morte celular causada pela Onco-A, e reforÃa que o composto pode ser um protÃtipo para o tratamento do cÃncer. / Auxemma oncocalyx Taub. belongs to the Boraginaceae family. It is known as âpau brancoâ and is frequently found in the State of CearÃ. The skin of that tree is an astringent and is commonly used as a medicine for wounds. Oncocalyxone A (Onco-A) is a quinone isolated from the ethanolic extract of A. oncocalyx, it has exhibited a series of pharmacological properties, such as analgesic, anti-inflammatory, antioxidant, cytotoxic and antitumor properties. The present study tried to provide a basic set of data on the cytotoxic effects of Onco-A against human promyelocytic leukemia cell line HL-60. The cytotoxic potential of Onco-A was evaluated by the MTT assay, colorimetric analysis, after 24 hours exposure in HL-60 cells with increasing concentrations (8, 16,5 e 33 ÂM). After the cells were exposure, there were performed experiments to identify the mecanisms of action in vitro (flow cytometry) by the cellular viability, phosphatidyl externalization, internucleosomal DNA fragmentation, identifying apoptotic pathway; the experiments to morphological analysis were made with tripan blue, May-Grunwald-Giemsa, and acridine orange/ethidium bromide; DNA integrityâs test by cometa assay; and the avaliation of interection of enzyme topoisomerase with Onco-A. Results: according to MTT test results, Onco-A showed a significant cytotoxic activity against HL-60 cells (IC50 11 ÂM). In addition, morphological analysis of cells treated with lower concentration of Onco â A demonstrated that there were reduction in cellâs volume, formation of apoptotic bodies, chromatin condensation, phosphatidylserine externalization, also reduction of cell viability and DNA fragmentation. The Onco-A caused mitochondrial depolarization, activated caspase starters -9 and -8, and the effectors -3 and -7, cleaved Poly ADP-Ribose polymerase â intrinsic and extrinsic pathways. Although our results had shown that none of the doses was able to generate Reactive Oxigen Species, after pre-treated for 1 hour with N-acetylcysteine, the cytotoxicity of Onco-A was changed, an increase in membrane integrity, decreased DNA fragmentation, drag on the G2/M cell cycle phase and low levels of DNA damage. Besides the Onco-A does not interact with the enzyme topoisomerase, the data suggest that their target as Michael acceptor, can be the DNA. These results suggest that apoptosis is one of the major forms of cellâs death caused by Onco-A, and reinforces the compound may be a prototype for cancer therapy.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / BenzotiazÃis sÃo compostos com anel benzeno ligados ao anel tiazol, que apresentam atividade antitumoral significante. Devido à alteraÃÃo de ligaÃÃes quÃmicas de molÃculas ser um mÃtodo comum para desenho de fÃrmacos em quÃmica medicinal e uma tÃcnica Ãtil para o desenvolvimento de novos medicamentos, a sÃntese de BenzotiazÃis substituÃdos apresentou muita vantagem para o tratamento de tipos especÃficos de cÃncer. No presente estudo foi avaliado o potencial citotÃxico de quatro anÃlogos de benzotiazÃis substituÃdos frente a linhagens tumorais e nÃo tumorais. Os resultados demonstraram que todos os anÃlogos apresentaram atividade citotÃxica, porÃm o composto 1 (CI50 = 1,65ÂM) foi escolhido para dar continuidade aos ensaios por ser protÃtipo dos demais, e o composto 3 (CI50 = 1,01ÂM) foi escolhido por apresentar maior citotoxicidade quando comparado aos demais e ainda por apresentar uma seletividade para linhagem nÃo tumoral avaliada, cÃlulas mononucleadas do sangue perifÃrico humano (CMSPH). Foi entÃo realizada uma sÃrie de ensaios in vitro, onde se buscou esclarecer os efeitos envolvidos nessa atividade citotÃxica. A linhagem HL60 foi a escolhida para dar continuidade ao restante dos experimentos por ser a linhagem mais sensÃvel à exposiÃÃo dos compostos. Nos ensaios de viabilidade foi observado uma reduÃÃo no nÃmero de cÃlulas viÃveis jà na concentraÃÃo de 1ÂM para o composto 1 (56,54% cÃlulas viÃveis, p<0,001) e para o composto 3 (38,45% cÃlulas viÃveis, p<0,001). A morfologia das cÃlulas HL-60 avaliadas atravÃs do uso da coloraÃÃo May-GrÃnwald-Giemsa revelou morte celular com caracterÃsticas de apoptose que ainda foi confirmado com ensaio da coloraÃÃo diferencial de laranja de acridina e brometo de etÃdio (LA/BE), onde foi considerada estatisticamente significante a partir da concentraÃÃo de 2ÂM do composto 1, com 79,92% (p<0,001) de cÃlulas com caracterÃsticas de apoptose e para o composto 3 na concentraÃÃo de 1ÂM com 28,86% (P<0,001). Nos ensaios de citometria de fluxo foi revelado que, quando tratados com os compostos, as cÃlulas HL60 promoviam a externalizaÃÃo da fosfatidil serina no qual na concentraÃÃo de 1ÂM foi considerado estatisticamente significante para o composto 1 (12,38% de cÃlulas em apoptose, p<0,001) e para o composto 3 (42,67%, p<0,001). Foi obervado ainda que os compostos ativam caspase 8 (via extrÃnseca), com 21,78% (P<0,001) de cÃlulas em apoptose para o composto 1 (2ÂM) e 47,5% (p<0,001) para o composto 3 (1ÂM); ativaram tambÃm caspase 9 (intrÃnseca), com 17,10% (p<0,001) de cÃlulas em apoptose para o composto 1 (1ÂM) e 33,55% (p<0,001) para o composto 3 (1(P<0,001)M); e conseqÃentemente ativaram as caspases 3 e 7, envolvidas no processo final da morte celular por apoptose, com 30,05% (p<0,001) de cÃlulas em apoptose para o composto 1 (2ÂM) e 53,19% (p<0,001) para o composto 3 (1ÂM). Foi observada intensa fragmentaÃÃo de DNA em cÃlulas tratadas com ambos os compostos, onde jà na concentraÃÃo de 1ÂM foi observado 23,13% (p<0,001, composto 1) e 61,02% (p<0,001, composto 3) de DNA fragmentado. Os composto nÃo foram capazes de induzir a formaÃÃo de espÃcies reativas de oxigÃnio (EROs) e ou causar danos diretos ou indiretos à fita de DNA. Conclui se que ambos podem ser considerados como molÃculas com potencial citotÃxico, destacando o composto 3 por sua maior citotoxicidade e seletividade. E ainda, esses dados reforÃam a importÃncia de sintetizar e estudar anÃlogos sintÃticos com atividades cada vez mais seletivas para o tratamento do cÃncer. / Benzothiazole are compounds consisted of a benzene ring with a thiazole ring that present significant anticancer activity, especially the fenil substituted. Due to molecular modeling, a common method for drug design in medicinal chemistry and a useful technique for the development of new drugs, the substituted synthesis of Benzothiazole presented great advantage for the treatment of specific types of cancer. In the present study it was evaluated the cytotoxic potential of four of synthetic benzothiazole analogues with malignant and non malignant cell lines. The results demonstrated that all the tested analogues presented cytotoxic activity, however compound 1 (CI50 = 1,65ÂM) was chosen to give continuity to the assays because it is the prototype of the others, and compound 3 (CI50 = 1,01ÂM) was chosen due to its higher cytotoxicity compared to the others and for presenting a selectivity towards malignant cell line when compared to a non malignant cell line, peripheral mononuclear blood cells(PMBC). A series of assays was then carried out in vitro, where it aimed to clarify effects involved in this cytotoxic activity. The cell line HL60 was chosen to conduct the experiments for it was the most sensitive cell line to the exposure of compounds. In the viability assay a reduction in the number of viable cells was observed in the concentration of 1ÂM for compound 1 (56.54% viable cells, p < 0.001) and compound 3 (38.45% viable cells, p< 0.001). The morphology of HL-60 cells evaluated with the use of the May-GrÃnwald-Giemsa dye showed cellular death with characteristics of apoptosis which was further confirmed with the orange acridine and ethidium bromide differential count assay (LA/BE), where it was considered statistically significant from the concentration of 2ÂM for compound 1, with 79,92% (p< 0.001) of cells with characteristics of apoptosis and for compound 3 in the concentration of 1ÂM with 28,86% (P< 0.001). In the flow cytometry assays it was observed that, when treated with the tested compounds, HL60 cells promoted the externalizaÃÃo of phosphatidyl serine in which in the concentration of 1ÂM it was considered statistically significant for compound 1 (12.38% of cells in apoptosis, p< 0.001) and for compound 3 (42.67%, p< 0.001). It was observed that the compounds activated caspase 8 (Extrinsic pathway), with 21,78% (P< 0.001) of cells with characteristics of apoptosis for compound 1 (2ÂM) and 47.5% (p< 0.001) for compound 3 (1ÂM); they also activated caspase 9 (intrinsic pathway), with 17,10% (p< 0.001) of cells with characteristics of apoptosis for compound 1 (1ÂM) and 33.55% (p< 0.001) for compound 3 (1ÂM); consequently, it was activated caspases 3 and 7, involved in the final process of apoptosis, with 30,05% (p< 0.001) of cells with characteristics of apoptosis for compound 1 (2ÂM) and 53.19% (p< 0.001) for compound 3 (1ÂM). Intense DNA fragmentation was observed in cells treated with the tested compounds, where in the concentration of 1ÂM it was observed 23.13% (p 0.001<, composition 1) and 61.02% (p 0.001<, composition 3) of fragmented DNA. The compounds were not able to generate reactive oxygen species (ROS) and cause direct or indirect damage to the DNA strand. In conclusion, both tested compounds can be considered as molecules with cytotoxic potential, highlighting compound 3 for its slightly higher cytotoxicity and selectivity. These data strengthen the importance to synthesize and to study synthetic compounds with more selective activities for the treatment of cancer.

This study reports the findings on the effect of Microcystin-LR (MCLR) on the gastrointestinal tract cells of mice and on two different cell lines, Caco2 and MCF-7. The cyanobacterium Microcystis aeruginosa produces the potent toxin, MCLR. This toxin has been implicated in a number of cases of ill-health. It was decided to investigate whether microcystin-LR induced apoptosis in the gastrointestinal tract of mice and also which possible mechanisms were involved in the induction in vitro. Balb/c mice were given a 75% LD50 intraperitoneal dose of pure microcystin -LR and sacrificed at 8, 16, 24 and 32 hours post-exposure. The small intestinal sections were stained with haematoxylin and eosin and examined for apoptotic cells. There was a time-dependent increase in the number of apoptotic cells with most in the duodenum and the jejunum. No change in glycogen content was evident at 24 hours post exposure when PAS-stained sections were examined. To determine that microcystin was the agent responsible for the changes, fluoroscein isothiocyanate (FITC) immunostaining for the toxin was done on the sections. Apoptosis in vitro was investigated in Caco2, a cell line that behaves like normal enterocytes when the cells are differentiated at confluency, and MCF-7, a breast cancer cell line deficient in pro-caspase-3, cells by 3-[dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and by staining with DAPI and Rhodamine 123. MCLR exposure induced apoptosis, as seen in decreased cell viability and increased leakage of LDH, as well as mitochondrial damage shown by Rhodamine staining. The MCF-7 cells, deficient in pro-caspase-3, and Caco2 cells did not show cleavage of poly(ADP)ribose polymerase (PARP) after exposure to 50μM MCLR after 72 hours exposure. Both micro- and milli-calpain activity was however significantly increased in both cell lines exposed to the toxin. There was a significant increase in H2O2, one of the key reactive oxygen species, production during the first 30 minutes that the cells were exposed to 50 mM MCLR.

>Magister Scientiae - MSc / Myocardial infarction, commonly known as a heart attack, is a condition where the blood supply to the heart tissue is cut off, starving the tissue from oxygen and nutrient supply, with consequent lethal damage to the heart tissue. This damage is as a result of the death of cardiomyocytes. Numerous studies demonstrated that the death of these cells is as a result of programmed cell death or apoptosis. Heat shock proteins can protect cardiomyocytes against cell death by inhibiting apoptosis. For this reason heat shock responsive proteins are emerging as therapeutic targets to suppress cell death in cardiomyocytes during myocardial infarction. RhoE and TIP41 are also amongst the genes that are upregulated in cardiomyocytes after heat stress. These genes do not encode classical heat shock proteins. The question that arises is whether the induction of RhoE during heat stress in cardiomyocytes has any cytoprotective role. This research project aims to investigate the potential cytoprotective role of RhoE and TIP41 in rat cardiomyocytes. Mutant cell lines that stably over-express RhoE and TIP41 were generated by transfecting H9c2 cells with the pcDNA-3.1-TOPO vector containing these genes. DNA transfections were performed using the metafectene transfection reagent. Over-expression was investigated using Western blot analysis. The mutant cell lines were treated with ceramide and camptothecin for a period of 24 hours and cell viability was assessed by the MTT assay. Two dimensional proteomic analysis was carried out to compare the proteomes of H9c2 and H9c2 cells that over-express RhoE. This research demonstrates that both RhoE and TIP41 are induced in response to heat stress and that the over-expression of RhoE is able to protect H9c2 against camptothecin induced cell death. Furthermore a proteomic 2D analysis demonstrates differential protein expression between H9c2 cells and H9c2 that over-express RhoE. Proteomic analysis demonstrates that the over-expression of RhoE leads to the down-regulation of Rho-GDI α. It can be concluded from this study that the expression of RhoE in response to heat shock is a cytoprotective event. The mechanism of cytoprotection is likely to involve Rho-GDI α.

Acute kidney injury (AKI), resulting from chemotherapeutic agents such as cisplatin, remains an obstacle in the treatment of cancer. Cisplatin-induced AKI involves apoptotic and necrotic cell death, pathways regulated by sphingolipids such as ceramide and glucosylceramide. Results from this study indicate that C57BL/6J mice treated with cisplatin had increased ceramide and hexosylceramide levels in the renal cortex 72 h following cisplatin treatment. Pretreatment of mice with inhibitors of acid sphingomyelinase and de novo ceramide synthesis (amitriptyline and myriocin, respectively) prevented accumulation of ceramides and hexosylceramide in the renal cortex and protected from cisplatin-induced AKI. To determine the role of ceramide metabolism to hexosylceramides in kidney injury, we treated mice with a potent and highly specific inhibitor of glucosylceramide synthase, the enzyme responsible for catalyzing the glycosylation of ceramides to form glucosylceramides. Inhibition of glucosylceramide synthase attenuated the accumulation of the hexosylceramides and exacerbated ceramide accumulation in the renal cortex following treatment of mice with cisplatin. Increasing ceramides and decreasing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI according to markers of kidney function, kidney injury, inflammation, cell stress, and apoptosis. Under conditions of high ceramide generation, data suggest that metabolism of ceramides to glucosylceramides buffers kidney ceramides and helps attenuate kidney injury.-Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Sharp, D. Siow, J. Megyesi, J. Shayman, A.Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury.

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