tacrolimus and 3-nitrotyrosine

Calcineurin inhibitors (cyclosporine and tacrolimus; CNIs) continue to be used as constituents of post-transplant immunosuppression in most centers. However, renal toxicity associated with the use of these drugs remains a problem adversely affecting the long-term graft survival. Fifteen adequate protocol renal allograft biopsies, with histological features of CNI toxicity among 140 protocol biopsies performed at 1-, 6-, and 12-month post-transplant, were included. Mitochondrial alterations in the tubular epithelial cells and endothelia of glomerular, peritubular capillaries and arterioles were graded semiquantitatively and further ultrastructural morphometric evaluation of numerical density and area of the mitochondria was performed. Immunohistochemical staining for nitrotyrosine (marker of peroxynitrite formation) and vascular endothelial growth factor (VEGF) was performed and expression graded semiquantitatively. Higher grades of alterations were seen in endothelial mitochondria as compared with tubular mitochondria in biopsies with calcineurin inhibitor toxicity (CNIT). Endothelial mitochondrial numerical density showed progressive decline over 1-, 6- and 12-month biopsies while area showed progressive increase in biopsies with CNIT as compared with controls. Upregulation of nitrotyrosine was seen even at 1-month post-transplant, persisted at 6 and 12 months, and was significantly greater than that in control biopsies. Intense VEGF expression was noted in early CNIT while progressive reduction was seen in 6- and 12-month protocol biopsies. This study shows a relatively high incidence of CNIT in protocol renal allograft biopsies, indicating that this might be an important mechanism of background damage to the allograft. Structural alterations in endothelial mitochondria are consistent findings in protocol biopsies with CNIT and this relatively specific mitochondrial damage may stem from the peroxynitrite-mediated damage associated with progressive loss of protective function of VEGF.

Topics: Adult; Biopsy; Calcineurin Inhibitors; Cyclosporine; Endothelium, Vascular; Female; Humans; Immunohistochemistry; Kidney Transplantation; Male; Mitochondria; Tacrolimus; Transplantation, Homologous; Tyrosine; Vascular Endothelial Growth Factor A

Immunophilin ligands and phosphodiesterase type 5 (PDE5) inhibitors are touted to promote erectile function recovery after cavernous nerve (CN) injury. However, the mechanisms for their effects remain unclear.. To compare the erection recovery effects of the immunophilin ligand FK506 and the PDE5 inhibitor sildenafil after CN injury and determine whether they involve antioxidative and/or antiapoptotic mechanisms.. Initial experiments established conditions of our CN injury model in adult male Sprague-Dawley rats. Subsequently, we evaluated treatment effects 14 days after: (i) unilateral CN injury (UNI) + saline (vehicle control); (ii) UNI + FK506 (5 mg/kg once daily, subcutaneous x 5 days); (iii) UNI + sildenafil (20 mg/kg every 8 hours, subcutaneous x 7 days); (iv) UNI + FK506/sildenafil; and (v) sham surgery.. Intracavernous pressure (ICP) measurement after CN electrical stimulation to assess erectile function and Western blot analysis of expressions of glutathione peroxidase (GPX; antioxidant enzyme), nitrotyrosine (NT; oxidative stress marker), and phosphorylated and total Akt (antiapoptotic factor) in penes.. In the UNI model, GPX expression was increased at Days 1 and 7, while p-Akt expression decreased at Day 1 and returned to baseline at Day 7. GPX expression was significantly higher in the UNI + FK506 group compared with the saline-treated group (P < 0.05). ICP increased in all treatment groups compared with that of the saline-treated group (P < 0.05). NT levels were increased after saline treatment (P < 0.05) but not after FK506 and sildenafil treatment, alone or in combination. GPX was localized to nerves coursing through the penis and to smooth muscle and endothelium of the dorsal vein and arteries.. Both FK506 and sildenafil protect erectile function after CN injury by decreasing oxidative stress-associated tissue damage. FK506 may act through increased GPX activity. Further research is required to elucidate mechanisms associated with the beneficial effect of sildenafil.

Topics: Animals; Blotting, Western; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Therapy, Combination; Glutathione Peroxidase; Immunohistochemistry; Male; Nerve Regeneration; Nitric Oxide Synthase; Penile Erection; Penis; Piperazines; Purines; Rats; Rats, Sprague-Dawley; Sildenafil Citrate; Sulfones; Tacrolimus; Tyrosine; Vasodilator Agents

Cyclosporin A (CsA) is known to preserve cardiac contractile function during endotoxemia, but the mechanism is unclear. Increased nitric oxide (NO) production and altered mitochondrial function are implicated as mechanisms contributing to sepsis-induced cardiac dysfunction, and CsA has the capacity to reduce NO production and inhibit mitochondrial dysfunction relating to the mitochondrial permeability transition (MPT).. We hypothesized that CsA would protect against endotoxin-mediated cardiac contractile dysfunction by attenuating NO production and preserving mitochondrial function.. Left ventricular function was measured continuously over 4 h in cats assigned as follows: control animals (n = 7); LPS alone (3 mg/kg, n = 8); and CsA (6 mg/kg, n = 7), a calcineurin inhibitor that blocks the MPT, or tacrolimus (FK506, 0.1 mg/kg, n = 7), a calcineurin inhibitor lacking MPT activity, followed in 30 min by LPS. Myocardial tissue was then analyzed for NO synthase-2 expression, tissue nitration, protein carbonylation, and mitochondrial morphology and function.. LPS treatment resulted in impaired left ventricular contractility, altered mitochondrial morphology and function, and increased protein nitration. As hypothesized, CsA pretreatment normalized cardiac performance and mitochondrial respiration and reduced myocardial protein nitration. Unexpectedly, FK506 pretreatment had similar effects, normalizing both cardiac and mitochondrial parameters. However, CsA and FK506 pretreatments markedly increased protein carbonylation in the myocardium despite elevated manganese superoxide dismutase activity during endotoxemia.. Our data indicate that calcineurin is a critical regulator of mitochondrial respiration, tissue nitration, protein carbonylation, and contractile function in the heart during acute endotoxemia.

Topics: Animals; Calcineurin Inhibitors; Cats; Cyclosporine; Endotoxemia; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Contraction; Myocardium; Nitric Oxide Synthase Type II; Peroxidase; Protein Carbonylation; Superoxide Dismutase; Tacrolimus; Tyrosine

The role of nitric oxide (NO) on tissue injury of hepatic allografts during rejection remains controversial. We investigated inducible nitric oxide synthase (iNOS) expression and formation of peroxynitrite in ACI rat liver grafts implanted in recipients. Animals were divided into four experimental groups: group I, isografts; group II, untreated hepatic allografts; group III, allografts treated with FK506; and group IV, allografts pretreated with donor-specific blood transfusion (DST). Serum nitrite/nitrate, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) concentrations increased significantly in group II rats after transplantation but were significantly lower in groups I, III, and IV. The numbers of macrophages that reacted with an antimacrophage iNOS monoclonal antibody as well as iNOS messenger RNA (mRNA) levels in liver specimens were also much lower in groups I, III, and IV as compared with group II. Immunostaining and Western blot analysis showed prominent tissue nitrotyrosine expression in untreated hepatic allografts, but not in allografts treated with FK506 or donor-specific blood. These results suggest that one of the mechanisms by which production of NO results in injury in rat hepatic allografts may be because of its reaction with superoxide to form peroxynitrite.

Topics: Animals; Blood Transfusion; Enzyme Inhibitors; Graft Rejection; Graft Survival; Liver Transplantation; Male; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; omega-N-Methylarginine; Rats; Rats, Inbred ACI; Rats, Inbred Lew; RNA, Messenger; Tacrolimus; Tissue Donors; Transplantation, Homologous; Transplantation, Isogeneic; Tyrosine