Inducción de Apoptosis in vitro en hepatocitos de rata por ciclosporina A

In rat hepatocytes and isolated liver mitochondrial fractions, CsA is often used as a specific inhibitor of mitochondrial Ca2+ release and as a specific blocker of mitochondrial membrane potential and permeability transition (MPT), which are all processes involved in the inhibition of apoptosis. However, neither inhibition nor induction of apoptosis by CsA has yet been described in the rat hepatocyte primary culture during incubation for 4 and 20 hours. It was the purpose of the present study to examine by means of morphological and biochemical criteria the effects of CsA on apoptosis, and to characterize the underlying mechanisms. Rat hepatocytes were cultured for 4 or 20 hours with CsA at concentrations of 0, 10, 25 and 50 μM. Chromatin condensation and fragmentation, DNA fragmentation (TUNEL), membrane phosphatidylserine distribution (Annexin V), caspase-1, -3 and -6 activity, mitochondrial membrane potential (Rhodamine 123), and cytochrome c release into the cytosol were investigated. Four hours after CsA treatment, chromatin condensation and fragmentation, and the number of TUNEL- and Annexin V-positive cells increased dose dependentlywithout any observable enzyme leakage, which indicated the integrity of the outer cell membrane. After 20 hours of CsA incubation apoptosis parameters were further increased and were accompanied by the increased activity of the cysteine protease, caspase-3 (CPP 32), and slightly increased caspase-6 (Mch 2), but not caspase-1 (ICE). The specific caspase-3 inhibitor, Ac-DEVD-CHO, inhibited caspase-3 activation and attenuated CsA-induced apoptosis and LDH leakage. The caspase-6 inhibitor, Ac-VEIDCHO, only marginally inhibited CsA-induced apoptosis. Decreased mitochondrial membrane potential and cytochrome c release went in parallel with ultrastructural mitochondrial changes and might be regarded as early events which trigger the apoptosis cascade. Transmission electron microscopy (TEM) confirmed an increase in the number of necrotic cells after 20 hours, but not after 4 hours, compared with controls.La cefalosporina A (CsA) se utiliza frecuentemente en hepatocitos de rata y fracciones mitocondriales hepáticas aisladas como inhibidor específico de la liberación de Ca2+ y como bloqueador selectivo de la permeabilidad y del potencial de membrana mitocondriales, procesos implicados en la inhibición de la apoptosis. Sin embargo, hasta ahora no se ha descrito ni la inhibición ni la inducción de apoptosis por CsA en cultivos primarios de hepatocitos de rata tras su incubación por un periodo de 4 a 20 horas. El propósito de este estudio ha sido examinar con criterios morfológicos y bioquímicos los efectos de la CsA sobre la apoptosis y esclarecer las características de los mecanismos subyacentes. Los hepatocitos de rata se cultivaron durante 4-20 h. con CsA a concentraciones de 0, 10, 25 y 50 μM. Se investigaron los fenómenos de condensación y fragmentación de la cromatina, fragmentación de ADN (TUNEL), distribución de fostatidilcolina en la membrana (Anexina V), así como la actividades enzimática de caspasas 1, -3 y –6, el potencial de membrana mitocondrial (Rhodamina 123) y la liberación de citocromo C en el citosol. Tras cuatro horas de incubación con CsA, la condensación y fragmentación de la cromatina y el número de células TUNEL y Anexina V positivas aumentaron en función de la dosis sin que se observara pérdida enzimática, lo que indica la integridad de la membrana celular externa. Después de 20 horas de incubación con CsA, experimentaron un mayor incremento acompañado del aumento de las actividad de cistein-proteasa, caspasa-3 (CPP32) y de un ligero incremento de caspasa-6 (Mch 2), pero no de caspasa-1 (ICE). El inhibidor específico de caspasa-3, Ac-DEVD-CHO,inhibió la activación de caspasa-3 y atenuó la apoptosis inducida por CsA y la pérdida de LDH. El inhibidor de caspasa-6, Ac-VEID-CHO, únicamente inhibió la apoptosis inducida por CsA. El descenso de potencial de membrana mitocondrial y la liberación de citocromo C fueron paralelos a los cambios de ultraestructuras mitocondriales y pudieran considerarse reacciones tempranas que desencadenan la cascada de fenómenos apoptóticos. La microscopia de transmisión electrónica (TEM) confirmó el incremento del número de células necróticas al cabo de 20 horas, pero no tras 4 horas de incubación, en comparación con los controles

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD

Penetration of ASM 981 in canine skin: a comparative study

ASM 981 has been developed for topical treatment of inflammatory skin diseases. It specifically inhibits the production and release of pro-inflammatory cytokines. We measured the skin penetration of ASM 981 in canine skin and compared penetration in living and frozen skin. To make penetration of ASM 981 visible in dog skin, tritium labelled ASM 981 was applied to a living dog and to defrosted skin of the same dog. Using qualitative autoradiography the radioactive molecules were detected in the lumen of the hair follicles until the infundibulum, around the superficial parts of the hair follicles and into a depth of the dermis of 200 to 500 microm. Activity could not be found in deeper parts of the hair follicles, the dermis or in the sebaceous glands. Penetration of ASM 981 is low in canine skin and is only equally spread in the upper third of the dermis 24 hours after application. Penetration in frozen skin takes even longer than in living canine skin but shows the same distribution

Effects of aliskiren on blood pressure, albuminuria, and (Pro)renin receptor expression in diabetic TG(mRen-2)27 Rats

The aim of this study was to explore the effects of the renin inhibitor aliskiren in streptozotocin-diabetic TG(mRen-2)27 rats. Furthermore, we investigated in vitro the effect of aliskiren on the interactions between renin and the (pro)renin receptor and between aliskiren and prorenin. Aliskiren distributed extensively to the kidneys of normotensive (non)diabetic rats, localizing in the glomeruli and vessel walls after 2 hours exposure. In diabetic TG(mRen-2)27 rats, aliskiren (10 or 30 mg/kg per day, 10 weeks) lowered blood pressure, prevented albuminuria, and suppressed renal transforming growth factor-β and collagen I expression versus vehicle. Aliskiren reduced (pro)renin receptor expression in glomeruli, tubules, and cortical vessels compared to vehicle (in situ hybridization). In human mesangial cells, aliskiren (0.1 μmol/L to 10 μmol/L) did not inhibit binding of I-renin to the (pro)renin receptor, nor did it alter the activation of extracellular signal-regulated kinase 1/2 by renin (20 nmol/L) preincubated with aliskiren (100 nmol/L) or affect gene expression of the (pro)renin receptor. Evidence was obtained that aliskiren binds to the active site of prorenin. The above results demonstrate the antihypertensive and renoprotective effects of aliskiren in experimental diabetic nephropathy. The evidence that aliskiren can reduce in vivo gene expression for the (pro)renin receptor and that it may block prorenin-induced angiotensin generation supports the need for additional work to reveal the mechanism of the observed renoprotection by this renin inhibitor