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.

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.

The role of growth factors on renal tubular cells submitted to hypoxia and deprived of glucose.

BACKGROUND: In acute renal failure (ARF) renal tubular cell death and detachment can be induced by necrotic and apoptotic mechanisms. Several studies have demonstrated some benefits of the use of growth factors in experimental models of ARF. METHODS: MDCK cells were cultured in a glucose-free medium for 24h and were submitted to hypoxia (PO2 around 35 mmHg) for additional 24 h. To evaluate the possible protective role of growth factors, EGF, IGF-I or HGF were added to the medium (20 ng mL). LDH release, viability (acridine orange and ethidium bromide dyes) and quantification of apoptotic cells (Hoechst 33342 dye fluorescence) were determined. RESULTS: In the injury group, an increase on LDH release (60% vs. 3%) and on number of apoptotic cells (22% vs. 0.2%) which was associated with a reduced cell viability (61% vs. 94%) when compared with controls. Only HGF, not EGF or IGF-I, was able to protect cells from injury. HGF caused a significant reduction on LDH release (30%) and on number of apoptotic cells (5%), with an increase on viability cellular (79%). CONCLUSIONS: HGF decreases cell death on MDCK cells after hypoxic-induced injury, probably acting in both necrotic and apoptotic mechanisms.

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/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.

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 Säo Paulo/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 percent, 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

Mecanismos celulares e moleculares da nefrotoxicidade pela cisplatina / Cellular and molecules mechanism of the nephrotoxicity at cisplatin

A cisplatina é um antineoplástico amplamente utilizado na quimioterapia de tumores, aumentando a sobrevida ou mesmo promovendo a cura do paciente. O seu principal efeito colateral é a nefrotoxicidade. Os mecanismos desse efeito adverso ainda näo säo bem conhecidos, podendo estar relacionados à apoptose, necrose, peroxidaçäo lipídica e aumento da concentraçäo de cálcio intracelular. A presente revisäo tem por objetivo colaborar para o conhecimento dessa faceta da cisplatina, condiçäo imprescindível para a formulaçäo de esquemas terapêuticos que atenuem a lesäo renal, sem comprometer a eficácia dessa importante droga.(au)

Nefrotoxicidade e morte celular (apoptose ou necrose) induzida pela cisplatina ao longo do néfron / Nefrotoxicitity and celular death (apoptosis or necrosis) induced by cisplatin along or nephron

A nefrotoxicidade é o fator limitante do antineoplásico cisplatina. Pelo fato de que a cisplatina afeta porções distintas do néfron, investigamos o efeito de duas concentrações de cisplatina (l ou lOOmM) em quatro linhagens diferentes de células: células mesangiais (CMI), células tubulares proximais (LLC-PKl), células tubulares distais (MDCK) e células de ducto coletor de medula interna (IMCD). O corante fluorescente para núcleos Hoechst 33342 foi empregado para quantificar as taxas de apoptose ( por cento), a eletroforese de DNA em gel de agarose, o método de TUNEL e a citometria de fluxo foram também utilizados. O método de exclusão utilizando acridina orange e brometo de etídeo foi utilizado para avaliar a viabilidade celular ( por cento). A viabilidade das células CMI diminuiu com a cisplatina 100 mM após 24 h (43,7ñ5,1/ 24 h; médiañEPM, *p