sirolimus and tempol

Topics: AMP-Activated Protein Kinases; Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Creatine Kinase, MM Form; Cyclic N-Oxides; Electron Transport Complex IV; GTPase-Activating Proteins; Heart Diseases; Mechanistic Target of Rapamycin Complex 1; Mice; Muscular Diseases; Myocardium; Oxidative Stress; Peptide Initiation Factors; Polyesters; Ribosomal Protein S6 Kinases; Sequestosome-1 Protein; Sirolimus; Spin Labels; Succinate Dehydrogenase; Superoxides; Tuberous Sclerosis Complex 1 Protein; Ubiquitinated Proteins

Inhibitors of the mammalian target of rapamycin (mTORi) can produce de novo proteinuria in kidney transplant patients. On the other hand, mTORi has been shown to suppress disease progression in several animal models of kidney disease. In the present study, we investigated whether glomerular permeability can be acutely altered by the mTORi temsirolimus and whether mTORi can affect acute puromycin aminonucleoside (PAN) or angiotensin II (ANG II)-induced glomerular hyperpermeability. In anesthetized Wistar rats, the left ureter was cannulated for urine collection, while simultaneously blood access was achieved. Temsirolimus was administered as a single intravenous dose 30 min before the start of the experiments in animals infused with PAN or ANG II or in nonexposed animals. Polydispersed FITC-Ficoll-70/400 (molecular radius 10-80 Å) and (51)Cr-EDTA infusion was given during the whole experiment. Measurements of Ficoll in plasma and urine were performed sequentially before the temsirolimus injection (baseline) and at 5, 15, 30, 60, and 120 min after the start of the experiments. Urine and plasma samples were analyzed by high-performance size-exclusion chromatography (HPSEC) to assess glomerular sieving coefficients (θ) for Ficoll10-80Å. Temsirolimus per se increased baseline glomerular permeability to Ficoll50-80Å 45 min after its administration, a reactive oxygen species (ROS)-dependent phenomenon. PAN caused a rapid and reversible increase in glomerular permeability, peaking at 5 min, and again at 60-120 min, which could be blocked by the ROS scavenger tempol. mTORi abrogated the second permeability peak induced by PAN. However, it had no effect on the immediate ANG II- or PAN-induced increases in glomerular permeability.

Topics: Angiotensin II; Animals; Cell Membrane Permeability; Cyclic N-Oxides; Glomerular Filtration Rate; Kidney Glomerulus; Male; Models, Animal; Puromycin Aminonucleoside; Rats; Rats, Wistar; Sirolimus; Spin Labels; Time Factors; TOR Serine-Threonine Kinases

To evaluate the effects of physiological levels of mitochondrial-derived reactive oxygen species (ROS) on skeletal muscle autophagy, a proteolytic pathway designed to regulate contractile and myofilament homeostasis and to recycle long-lived proteins and damaged organelles.. Basal levels of autophagy and autophagy triggered by 1.5 to 4 h of acute nutrient deprivation, rapamycin treatment, or leucine deprivation were measured in differentiated C2C12 myotubes using long-lived protein degradation assays, LC3B lipidation, autophagy-related gene expression, and electron microscopy. Preincubation with the general antioxidants tempol (superoxide dismutase mimic) and N-acetyl cysteine (NAC) or the mitochondria-specific antioxidants mito-tempol and SS31 significantly decreased the rates of long-lived protein degradation and LC3B flux and blocked the induction of autophagy-related gene expression. Mitochondrial ROS levels significantly increased in response to acute nutrient deprivation and rapamycin treatment. Mito-tempol and tempol blocked this response. Antioxidants decreased AMP-activated protein kinase (AMPK) phosphorylation by 40% and significantly increased protein kinase B (AKT) phosphorylation, but exerted no effects on mTORC1-dependent ULK1 phosphorylation on Ser(555). NAC significantly decreased basal LC3B autophagic flux in skeletal muscles of mice.. We report for the first time that endogenous ROS promote skeletal muscle autophagy at the basal level and in response to acute nutrient starvation and mTORC1 inhibition. We also report for the first time that mitochondrial-derived ROS promote skeletal muscle autophagy and that this effect is mediated, in part, through regulation of autophagosome initiation and AKT inhibition.. Mitochondrial-derived ROS promote skeletal muscle autophagy and this effect is mediated, in part, through activation of AMPK and inhibition of AKT.

Topics: AMP-Activated Protein Kinases; Animals; Antioxidants; Autophagy; Cell Line; Cyclic N-Oxides; Food; Mice; Mitochondria; Muscle Fibers, Skeletal; Muscle, Skeletal; Phosphorylation; Proteolysis; Reactive Oxygen Species; Signal Transduction; Sirolimus; Spin Labels

Rapamycin, a widely used immunosuppressive drug, has been shown to interfere with the function of dendritic cells (DCs), antigen-presenting cells contributing to the initiation of primary immune responses and the establishment of immunological memory. DC function is governed by the Na(+)/H(+) exchanger (NHE), which is activated by bacterial lipopolysaccharides (LPS) and is required for LPS-induced cell swelling, reactive oxygen species (ROS) production and TNF-α release. The present study explored, whether rapamycin influences NHE activity and/or ROS formation in DCs. Mouse DCs were treated with LPS in the absence and presence of rapamycin (100 nM). ROS production was determined from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, and TNF-α production utilizing ELISA. In the absence of LPS, rapamycin did not significantly modify cytosolic pH, NHE activity or cell volume but significantly decreased ROS formation. LPS stimulated NHE activity, enhanced forward scatter, increased ROS formation, and triggered TNF-α release, effects all blunted in the presence of rapamycin. NADPH oxidase inhibitor Vas-2870 (10 μM) mimicked the effect of rapamycin on LPS induced stimulation of NHE activity and TNF-α release. The effect of rapamycin on TNF-α release was also mimicked by the antioxidant ROS scavenger Tempol (30 μM) and partially reversed by additional application of tert-butylhydroperoxide (10 μM). In conclusion, in DCs rapamycin disrupts LPS induced ROS formation with subsequent inhibition of NHE activity, cell swelling and TNF-α release.

Topics: Animals; Benzoxazoles; Cell Size; Cells, Cultured; Cyclic N-Oxides; Cytosol; Dendritic Cells; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Flow Cytometry; Fluoresceins; Fluorescence; Hydrogen-Ion Concentration; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Reactive Oxygen Species; Sirolimus; Sodium-Hydrogen Exchangers; Spin Labels; tert-Butylhydroperoxide; Triazoles; Tumor Necrosis Factor-alpha