The effect of an HIV-1 viral protease inhibitor on staurosporine-induced apoptosis in immortalized mesangial cells

CONTEXT: Progressive glomerular sclerosis is a condition characterized by the accumulation of glomerular extracellular matrix and a decrease in the number of glomerular cells. The mechanisms involved in the progressive loss of glomerular cells are not well understood but may involve the process of apoptosis. The principal mediators for the apoptotic pathway are a class of protease enzymes called caspases. It is not known how other therapeutic protease inhibitors affect the caspase cascade and therefore whether they would be effective in preventing excessive apoptosis in the late stages of progressive glomerular sclerosis. OBJECTIVE: To evaluate whether an inhibitor of the HIV-1 viral protease Ac-Leu-Val-phenylalanine (PI) could inhibit apoptosis in immortalized mesangial cells. DESIGN: Experimental. SETTING: Nephrology Division, Universidade Federal de São Paulo (UNIFESP)/Escola Paulista de Medicina. PARTICIPANTS: Immortalized mesangial cells. PROCEDURES: Cell culture. MAIN MEASUREMENTS: Viability and rate of apoptosis. RESULTS: Immortalized mesangial cells were treated with staurosporine (at concentrations of 10-100 nM for 8-28 hours) to induce apoptosis. Staurosporine at 10 nM for 8 hours had no effect on viability, but did cause a significant increase in the rate of apoptosis (p = 0.0411, n = 6). Increasing the incubation time elicited a greater increase in the rate of apoptosis (p = 0.0001, n = 6), although there was also a significant decrease in viability (p=0.0002). Increasing the concentration of staurosporine to 100 nM resulted in a marked increase in apoptosis (p <0.0001) but resulted in unacceptable viability (<40%, p <0.0001, n = 6). CONCLUSIONS: Incubation of immortalized mesangial cells with PI (900 nM) alone for 2-24 hours had no effect on cell viability or the rate of apoptosis when compared with vehicle (methanol) controls. Co-incubation of the cells with staurosporine (10 nM) and PI for 24 hours had no significant effect on the rate of apoptosis. Therefore, in immortalized mesangial cells, staurosporine-induced apoptosis was not significantly affected by the HIV-1 viral protease inhibitor Ac-Leu-Val-phenylalanine.CONTEXTO: A glomeruloesclerose progressiva (GEP) é uma situação caracterizada pela acumulação de matriz extracelular glomerular e pela diminuição no número de células no glomérulo. Os mecanismos envolvidos na perda progressiva do número glomerular da célula não são bem compreendidos, mas podem envolver o processo da apoptose. Os principais mediadores na evolução da apoptose são uma classe de enzimas de protease chamadas caspases. Não se sabe como outros inibidores de proteases com potencial uso terapêuticos efetuam a cascata dos caspases e, conseqüentemente, se podem ser eficazes em impedir a apoptose nos estágios anteriores da glomeruloesclerose progressiva. OBJETIVO: Avaliar se um inibidor da protease HIV-1 viral (Ac-Leu-Val-phenylalanine; o PI) poderia prevenir a apoptose em células mesangiais glomerulares imortalizadas. TIPO DE ESTUDO: Estudo experimental LOCAL: Disciplina de Nefrologia da Universidade Federal de São Paulo (UNIFESP)/Escola Paulista de Medicina, São Paulo, Brasil. PARTICIPANTES: Células Mesangiais Imortalizadas. PROCEDIMENTOS: Cultura de Célula. VARIÁVEIS ESTUDADAS: Viabilidade e taxa de apoptose. RESULTADOS: As células mesangiais imortali zadas foram tratadas com a estaurosporina (10-100 nM por 8 a 28h) para induzir a apoptose. A estaurosporina (10 nM; 8 h) não teve nenhum efeito viável, mas causou um significativo aumento na taxa de apoptose (p = 0,0411, n = 6). O aumento do tempo de incubação proporcionou um grande aumento da taxa de apoptose (p = 0,0001, n = 6), entretanto, há também uma significativa diminuição da viabilidade (p = 0,0002). O aumento da concentração de estaurosporina 100 nM resultou em aumento da apoptose (p <0,0001), porém com níveis inaceitáveis de viabilidade (< 40%, p <0,0001, n = 6). CONCLUSÕES: A incubação de células mesangiais imortalizadas com PI (900 nM) isolado por 2-24 h não teve efeito na viabilidade celular nem na taxa de apoptose comparada aos meios de controle (metanol). Co-incubação das células com estaurosporina (10 nM) e PI por 24 horas não adicionou efeito significativo na taxa de apoptose. Conseqüentemente, nas células mesangiais imortalizadas a apoptose induzida por estaurosporina não foi afetada significativamente pelo inibidor viral da protease HIV-1 Ac-Leu-Val-phenylalanine.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Department of MedicineUNIFESP, EPM, Department of MedicineSciEL

Loss of LAT1 sex-dependently delays recovery after caerulein-induced acute pancreatitis

Background: The expression of amino acid transporters is known to vary during acute pancreatitis (AP) except for LAT1 (slc7a5), the expression of which remains stable. LAT1 supports cell growth by importing leucine and thereby stimulates mammalian target of rapamycin (mTOR) activity, a phenomenon often observed in cancer cells. The mechanisms by which LAT1 influences physiological and pathophysiological processes and affects disease progression in the pancreas are not yet known. Aim: To evaluate the role of LAT1 in the development of and recovery from AP. Methods: AP was induced with caerulein (cae) injections in female and male mice expressing LAT1 or after its knockout (LAT1 Cre/LoxP). The development of the initial AP injury and its recovery were followed for seven days after cae injections by daily measuring body weight, assessing microscopical tissue architecture, mRNA and protein expression, protein synthesis, and enzyme activity levels, as well as by testing the recruitment of immune cells by FACS and ELISA. Results: The initial injury, evaluated by measurements of plasma amylase, lipase, and trypsin activity, as well as the gene expression of dedifferentiation markers, did not differ between the groups. However, early metabolic adaptations that support regeneration at later stages were blunted in LAT1 knockout mice. Especially in females, we observed less mTOR reactivation and dysfunctional autophagy. The later regeneration phase was clearly delayed in female LAT1 knockout mice, which did not regain normal expression of the pancreas-specific differentiation markers recombining binding protein suppressor of hairless-like protein (rbpjl) and basic helix-loop-helix family member A15 (mist1). Amylase mRNA and protein levels remained lower, and, strikingly, female LAT1 knockout mice presented signs of fibrosis lasting until day seven. In contrast, pancreas morphology had returned to normal in wild-type littermates. Conclusion: LAT1 supports the regeneration of acinar cells after AP. Female mice lacking LAT1 exhibited more pronounced alterations than male mice, indicating a sexual dimorphism of amino acid metabolism

Nefrotoxicitity and celular death (apoptosis or necrosis) induced by cisplatin along or nephron

A nefrotoxicidade e o fator limitante do antineoplasico cisplatina. Pelo fato de que a cisplatina afeta porcoes distintas do nefron, investigamos o efeito de duas concentracoes de cisplatina (l ou lOOmM) em quatro linhagens diferentes de celulas: celulas mesangiais (CMI), celulas tubulares proximais (LLC-PKl), celulas tubulares distais (MDCK) e celulas de ducto coletor de medula interna (IMCD). O corante fluorescente para nucleos Hoechst 33342 foi empregado para quantificar as taxas de apoptose ( por cento), a eletroforese de DNA em gel de agarose, o metodo de TUNEL e a citometria de fluxo foram tambem utilizados. O metodo de exclusao utilizando acridina orange e brometo de etideo foi utilizado para avaliar a viabilidade celular ( por cento). A viabilidade das celulas CMI diminuiu com a cisplatina 100 mM apos 24 h (43,7n5,1/ 24 h; medianEPM, *p<O,O5, n=4). No entanto, a taxa de apoptose nao foi modificada, sugerindo assim necrose. Com a cisplatina lmM, as taxas de apoptose aumentaram apos 72 h (l3,8n3,4 por cento*) e a viabilidade nao mudou. Nas celulas LLC-PKL e MDCK, a apoptose aumentou com cisplatina 100 mM apos 24 horas (LLC-PKl: 82,0n5,7 por cento* e MDCK: 93,5n4,4 por cento*) e novamente, a viabilidade nao foi modificada. Com a cisplatina lmM a apoptose e a viabilidade nao foram modificadas. As celulas IMCD com cisplatina 100 mM apos 48 h tiveram a sua viabilidade diminuida (28,0n4,1 por cento*), a taxa de apoptose aumentou, mas menos que nas celulas LLC-PKL e MDCK (27,9n7,1 por cento/24h*). Com cisplatina l mM apos 72 h houve aumento da apoptose (20,0n6,7 por cento*). Apos determinar que a morte foi tipo, concentracao e tempo dependente, analisamos o mecanismo molecular envolvido nas celulas LLC-PKL e MDCK. Observamos que o inibidor da caspase-3 (z-DEVD-fmk) reduziram as taxas de apoptose em 36 por cento nas celulas LLC-PKL (l6OmM) e 91 por cento nas MDCK (l6mM). A expressao da proteina p53 foi aumentada em 12 por cento nas MDCK e diminuiu 21 por cento nas LLC-PKL tratadas com cisplatina quando analisadas pelo Western blot. O envolvimento da concentracao do calcio intracelular ([Ca 2+]i) e da peroxidacao lipidica no mecanismo de morte foram avaliadas. cisplatina nao mudou a [Ca2+]i quando analisada fluorimetricamente, assim como o bloqueador de canais de calcio, nifedipina, nao mudou as taxas de apoptose nas celulas tratadas com cisplatina. Os niveis de peroxidacao lipidica tambem nao foram modificados. Em conclusao...(au)BV UNIFESP: Teses e dissertaçõe

Evaluation of the effect of selenium on cisplatin-induced nephrotoxicity in rats

A cisplatina é um antineoplásico muito utilizado em clínica oncológica para o tratamento de câncer de ovário, testículo e bexiga, entre outros. Um dos fatores limitantes de seu uso é a nefrotoxicidade dose-dependente induzida pela droga. O mecanismo dessa nefrotoxicidade ainda é desconhecido, e a peroxidação lipídica parece estar envolvida neste processo. Várias substâncias têm sido utilizadas para tentar reduzir a nefrotoxicidade da cisplatina. O objetivo deste trabalho foi avaliar o efeito do selênio (administrado por via oral) na nefrotoxicidade induzida pela cisplatina (administrada por via intraperitoneal) em ratos Wistar. Os animais foram divididos em 4 grupos, Controle (n=6), Selênio (n=6), Cisplatina (n=6) e Cisplatina+Selênio (n=6). Sete dias após a administração da cisplatina foram avaliados os níveis de peroxidação lipídica pelo método das substâncias reativas ao ácido tiobarbitúrico e os níveis de glutationa nos rins. Para avaliar o efeito nefrotóxico determinou-se os níveis diários da enzima N-acetil-p-D-glucosaminidase (NAG) total e isoenzima B, volume urinário, proteína urinária total, creatinina plasmática e \"clearance\" da creatinina. Podemos sugerir que a peroxidação lipídica parece estar envolvida mecanismo de nefrotoxicidade da cisplatina. Os resultados de peso corporal e dos rins, creatinina e proteinúria obtidos, permitem concluir que o selênio administrado por gavagem 24 horas antes de uma dose única de cisplatina, administrada intraperitonealmente, não protegeu os ratos da nefrotoxicidade induzida pela cisplatina. Porém se avaliarmos os resultados de volume urinário e isoenzima B da NAG podemos sugerir que o selênio promoveu uma proteção parcial contra as lesões precoces induzida nos túbulos proximais pela cisplatina.Cisplatin is an important antineoplastic drug, widely used against ovarian, testis and bladder câncer. Nephrotoxicity has been observed as a dose-limiting factor in cisplatin therapy. The mechanism of cisplatin-induced nephrotoxicity is not know yet and it have been associated with renal lipidic peroxidation. Administration of some substances has been used to protect against the nephrotoxicity. The aim of this study was to determine whether selenium (sodium selenite, by gavage) provide protection against cisplatininduced nephrotoxicity in Wistar rats. Animais were divided in 4 groups (n=6), wich were treated with i.p. injections of cisplatin 5mg/kg (Cisplatin and Cisplatin+selenium groups). The influence of selenium on cisplatin nephrotoxicity was studied by oral administration of sodium selenite 24 hours prior to the cisplatin administration. After 7 days of cisplatin administration, the rats were sacrifíed and the renal leveis of lipid peroxidation were evaluated by the method of tiobarbituric acid reactive substances and renal glutathion. The daily levei of urinary N-acetyl-p-Dglucosaminidase total and isoenzyme B, urinary volume, total urinary protein, plasmatic creatinine and creatinine clearance were determined to evaluate the nephrotoxicity. We can suggest that lipidic peroxidation may be associated with cisplatin-induced nephrotoxicity mecanism. Oral administraiion of selenium did not provide protection against cisplatininduced nephrotoxicity, due the body weight, kidney weight, plasmatic creatinine, creatinine clearance, and total protein urinaiy results. However considering urinary volume and B isoenzyme activity results, we can suggest that selenium provides partial protection to early injury cisplatin-induced in proximal tubules

Mucosal Monosaccharide Transporter Expression in Newborns With Jejunoileal Atresia and Along the Adult Intestine

OBJECTIVES: In newborn rodents, intestinal maturation involves delayed fructose transporter GLUT5 expression until weaning. In jejunoileal atresia (JIA), distal intestinal segments lack exposure to amniotic fluid-containing carbohydrates. We assessed in human newborns, the impact of intestinal maturation and obstruction on mucosal monosaccharide transporter expression. METHODS: Samples were obtained from 10 newborns operated for small intestinal atresia and from 17 adults undergoing gastroduodenoscopy and/or ileocolonoscopy. mRNA expression of the transporters SGLT1, GLUT1, GLUT2, GLUT5, and GLUT7 was measured in neonate samples proximal and distal of the atresia as well as in adult duodenum, ileum, and colon. Protein expression and localization was assessed using immunofluorescence. RESULTS: Although mRNA expression of monosaccharide transporters did not significantly differ between newborn and adult samples, luminal fructose transporter GLUT5 protein was absent in 0- to 4-day-old neonates, but expressed in adults. The mRNA expression of the 5 tested monosaccharide transporters was unchanged distal from the JIA relative to proximal. Similarly, luminal sodium-dependent glucose transporter SGLT1 and basolateral GLUT2 were expressed proximal and distal to JIA as visualized by immunofluorescence staining. With the exception of glucose transporter GLUT1 that showed highest expression levels in colon, all investigated hexose transporters showed strongest expression in duodenum, lower levels in ileum and lowest in colon. CONCLUSIONS: Human newborns lack small intestinal fructose transporter GLUT5 protein expression and small intestinal atresia does not affect the expression of hexose transporters