FK506 Pretreatment Research Articles

Cascading renal injury after brain death: Unveiling glycocalyx alteration and the potential protective role of tacrolimus.

Brain death (BD) is a complex medical state that triggers systemic disturbances and a cascade of pathophysiological processes. This condition significantly impairs both kidney function and structural integrity, thereby presenting considerable challenges to graft viability and the long-term success of transplantation endeavors. Tacrolimus (FK506), an immunosuppressive drug, was used in this study to assess its impact as a pretreatment on brain death-induced renal injury. This study aimed to investigate changes associated with brain death-induced renal injury in a 4-month-old female porcine model. The experimental groups included brain death placebo-pretreated (BD; n = 9), brain death tacrolimus-pretreated using the clinical dose of 0.25mg/kg the day before surgery, followed by 0.05mg/kg/day 1hour before the procedure (BD + FK506; n = 8), and control (ctrl, n = 7) piglets, which did not undergo brain death induction. Furthermore, we aimed to assess the effect of FK506 on these renal alterations through graft preconditioning. We hypothesized that immunosuppressive properties of FK506 reduce tissue inflammation and preserve the glycocalyx. Our findings revealed a series of interconnected events triggered by BD, leading to a deterioration of renal function and increased proteinuria, increased apoptosis in the vessels, glomeruli and tubules, significant leukocyte infiltration into renal tissue, and degradation of the glycocalyx in comparison with ctrl group. Importantly, treatment with FK506 demonstrated significant efficacy in attenuating these adverse effects. FK506 helped reduce apoptosis, maintain glycocalyx integrity, regulate neutrophil infiltration, and mitigate renal injury following BD. This study offers new insights into the pathophysiology of BD-induced renal injury, emphasizing the potential of FK506 pretreatment as a promising therapeutic intervention for organ preservation, through maintaining endothelial function with the additional benefit of limiting the risk of rejection.

Sevoflurane induces neurotoxic effects on developing neurons through the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway.

Children repeatedly exposed to anaesthesia have a high risk of cognitive impairment, but the mechanism of its regulation in this context is unknown. The objective of this study was to investigate the possible toxic mechanism of sevoflurane through the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway. The hippocampal neuronal HT22 cell line was used in this study. The intervention group was treated with the WNK1 inhibitor WNK-463, CaN inhibitor FK506 and Drp-1 inhibitor Mdivi-1 respectively in the medium for 30 min before sevoflurane anaesthesia. The sevofluane group and all intervention group treated with 4.1% sevoflurane for 6h. Compared with the control group, sevoflurane treatment decreased cell viability and increased cellular apoptosis. Our study found that WNK-463, FK506 and Mdivi-1 can all alleviate the sevoflurane-induced reduction in cell viability, decrease the cell apoptosis. In addition, WNK-463 pretreatment could inhibit the increase of WNK1 kinase and NKCC1 protein concentration caused by sevoflurane. Further, sevoflurane anaesthesia causes intracellular calcium overload, increases the expression of CaN and induces the dephosphorylation of Drp-1 protein at ser637, while CaN inhibitor FK506 pretreatment could reduce the dephosphorylation of Drp-1. Therefore, the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway plays an important role in sevoflurane-related neurotoxicity. Reducing intracellular calcium influx may be one of the important mechanism to ameliorate sevoflurane toxicity.

Neuroinflammation-mediated mitochondrial dysregulation involved in postoperative cognitive dysfunction

Neuroinflammation following peripheral surgery is a pivotal pathogenic mechanism of postoperative cognitive dysfunction (POCD). However, the key site of inflammation-mediated neural damage remains unclear. Impaired mitochondrial function is a vital feature of degenerated neurons. Dynamin-related protein 1 (DRP1), a crucial regulator of mitochondrial dynamics, has been shown to play an essential role in synapse formation. Here, we designed experiments to assess whether Drp1-regulated mitochondrial dynamics and function are involved in the pathological processes of POCD and elucidate its relationship with neuroinflammation. Aged mice were subjected to experimental laparotomy under isoflurane anesthesia. Primary neurons and SH-SY5Y cells were exposed to tumor necrosis factor (TNF). We found an increase in Drp1 activation as well as mitochondrial fragmentation both in the hippocampus of mice after surgery and primary neurons after TNF exposure. Pretreatment with Mdivi-1, a Drp1 specific inhibitor, reduced this mitochondrial fragmentation. Drp1 knockdown with small interfering RNA blocked TNF-induced mitochondrial fragmentation in SH-SY5Y cells. However, the application of Mdivi-1 exhibited a negative impact on mitochondrial function and neurite growth in primary neurons. Calcineurin activity was increased in primary neurons after TNF exposure and contributed to the Drp1 activation. The calcineurin inhibitor FK506 exhibited a Drp1-independent function that mitigated mitochondrial dysfunction. Finally, we found that FK506 pretreatment ameliorated the neurite growth in neurons treated with TNF and the learning ability of mice after surgery. Overall, our research indicated a crucial role of mitochondrial function in the pathological processes of POCD, and neuronal metabolic modulation may represent a novel and important target for POCD.

Pre-treatment with FK506 reduces hepatic ischemia-reperfusion injury in rats

The study is aimed to investigate the protective effects and possible mechanism of tacrolimus (FK506) pre-treatment in hepatic ischemia-reperfusion injury in rats. The rats were randomly assigned into four groups, which were S, IR, L and H group, and then all groups were subjected to 60min of 70% partial warm liver ischemia, except S group. Rats in the L and H group were pre-treated with two different doses FK506 at 60min before ischemia. The rats of the IR group received an identical volume of normal saline. All animals were sacrificed after 6h of reperfusion. Transaminases were measured by biochemistry analyzer. Elisa kit was used to detect TNF-α, IL-6 and IL-1β levels in serum. Liver specimens were stained with hematoxylin and eosin (HE) to assess the pathologic changes. The expressions of heme oxygenase-1 (HO-1), hypoxia-inducible factor-1α (HIF-1α), nuclear factor of activatedT cells (NFAT3) were measured by real-time quantitative PCR and western blotting and the Bcl-2 and the Bax protein were tested by western blotting. In rats pre-treated with FK506, the levels of transaminases, TNF-α and IL-1β were reduced significantly and also liver damage was dramatically mitigated compared to those without FK506 pre-treatment. Moreover, the expression of HO-1 at the level of both transcription and translation increased clearly and the activation of the HIF-1α was found in FK506 pre-treated livers. Moreover, NFAT3 protein transportation to the nucleus was reduced and Bax protein expression was decreased, but the expression of Bcl-2 protein was markedly increased after FK506 pre-treatment. FK506 pre-treatment could lessen hepatic ischemia-reperfusion injury through up-regulating the expression of HIF-1α and HO-1, and inhibiting nuclear translocation of NFAT3 in liver tissues.

Tacrolimus pretreatment on liver ischemia reperfusion injury in rats

Objective To determine the effects of tacrolimus (FK506) pretreatment on the liver ischemia reperfusion (IR) injury. Methods 32 mature SD rats were randomly assigned into four groups, which were sham-operated group (S), ischemia reperfusion group (IR), low-dose FK506-treated group (L) and high-dose FK506-treated group (H). After the treatment of liver ischemia for 60 minutes and reperfusion for 6 hours, the levels of serum ALT and AST in rats were tested. The TNF-α and IL-1β levels were evaluated by enzyme linked immunosorbent assay. Liver damage was assessed by paraffin sections stained with H&E. The quantitative real-time PCR, the immunohistochemistry and Western blot were used to detect the expression of HMGB1 mRNA and protein with or without FK506 pretreatment. Results The levels of serum ALT [(424.0±137.4)U/L, (291.0±42.0)U/L], AST [(554.2±127.7)U/L, (410.2±7.0)U/L], TNF-α [(115.1±49.0) ng/L, 120.4±28.5) ng/L] and IL-1β [(424.5±105.2) ng/L, (612.1±49.6) ng/L] decreased markedly in the group L and group H compared with the group IR (P 0.05). Conclusion FK506 pretreatment can protect the liver by reducing the expression of HMGB1, inhibiting the release of inflammatory cytokines and alleviating cell necrosis after the liver ischemia reperfusion injury in rats. Key words: Tacrolimus; Liver; Ischemia reperfusion; High mobility group box 1 protein, HMGB1; Liver protection

Effect of the calcineurin inhibitor FK506 on K+–Cl− cotransporter 2 expression in the mouse hippocampus after kainic acid-induced status epilepticus

Calcineurin (CaN)-mediated excitotoxicity impairs γ-aminobutyric acid (GABA) transmission and induces neuronal apoptosis. Ca(2+)-dependent K(+)-Cl(-) cotransporter 2 (KCC2) participates in GABAergic inhibitory transmission. However, the mechanism by which CaN mediates GABA receptor-mediated KCC2 in seizures is not fully understood. In the present study, we investigated the altered expression of KCC2 and the effects of the CaN inhibitor FK506 on KCC2 expression in the mouse hippocampus following kainic acid (KA) treatment. FK506 was injected twice 24 h and 30 min before KA treatment and then mice were treated with KA and killed 2 days later. FK506 had anticonvulsant effect on KA-induced seizure activities. CaN cleavage was evident in the hippocampus 24 h after KA treatment. FK506 pretreatment blocked the truncation of CaN in the KA-treated hippocampus. Cresyl violet and TUNEL staining showed that FK506 prevented KA-induced hippocampal cell death. In particular, Western blot analysis showed that KCC2 expression was time dependent, with a peak at 6 h and a return to decreased levels at 48 h, whereas FK506 pretreatment inhibited the KA-induced decrease in KCC2 expression in the hippocampus. Immunofluorescence showed that FK506 pretreatment protected the loss of inhibitory GABAergic KCC2-expressing neurons following KA treatment. Taken together, these results provide evidence that altered KCC2 expression may be associated with Ca(2+)-mediated seizure activity and indicate that neuron-specific KCC2 may be involved in neuroprotection after seizures.

Calcineurin Regulates Myocardial Function during Acute Endotoxemia

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.

Failure of FK506 (tacrolimus) to alleviate apomorphine-induced circling in rat Parkinson model in spite of some cytoprotective effects in SH-SY5Y dopaminergic cells

The mechanism of action of the neurotoxin 6-hydroxydopamine (6-OHDA) is thought to involve the generation of free radicals and subsequent apoptotic processes. We have demonstrated in vitro that the neuroimmunophilin, FK506 (10–100 nM), dose dependently and significantly restored the ROS production to the control level, increased the Bcl-2 protein level, partly inhibited the cytochrome C release from mitochondria and reduced the caspase-3 activation in SH-SY5Y cells. On the other hand, there was no significant restoration of the ATP level by FK506 and the toxin activated proteins, p53 and Bax, were not normalized by FK506. In support of these latter results, daily administration of FK506 for 7 days to rats (0.5, 1 and 3 mg/kg i.p.) did not significantly prevent the apomorphine-induced contralateral circling, measured 2 weeks after unilateral nigral lesioning. Moreover, FK506 pretreatment did not significantly lower the toxin elevated lipid peroxidation levels, indicating that oxidative stress was present even after the FK506 treatment in the lesioned striatum. Taken together, our results with FK506 are inconsistent. We confirm the antioxidant nature of FK506, that is, it blocks ROS production in SH-SY5Y cells. However, there were no significant protective effects in any apoptotic analyses in SH-SY5Y cells and in animal studies, a 7-day FK506 pre-treatment was not able to reverse the toxic effect of 6-OHDA in a rat model of Parkinson's disease.

Immunophilin ligands FK506 and cyclosporine A improve neurologic and histopathologic outcome after transient spinal cord ischemia in rabbits

We comparatively evaluated the protective effect of the immunophilin ligands cyclosporine A (INN: ciclosporin), FK506, and rapamycin on the spinal cord in a rabbit model of transient ischemia. Both cyclosporine A and FK506 inhibit calcineurin, whereas rapamycin does not. Thirty-six male New Zealand White rabbits were divided into the following 6 groups: group C, 15 minutes of spinal cord ischemia; group FK, FK506 (1 mg/kg) administered 30 minutes before ischemia; group CsA, cyclosporine A (30 mg/kg) administered 30 minutes before ischemia; group CsA-C, chronic administration of cyclosporine A (20 mg/kg) for 9 days before ischemia; group R, rapamycin (1 mg/kg) administered 30 minutes before ischemia; and group R+FK, rapamycin (1 mg/kg) administered 20 minutes before FK506 pretreatment (1 mg/kg). Group CsA-C was added because the drug does not readily cross the blood-brain barrier. Neurologic function was evaluated by Johnson's 5-point scale at 8, 24, and 48 hours after ischemia, and histopathology was assessed 48 hours after ischemia. At 24 and 48 hours after ischemia, the Johnson score was better in groups FK (4.0 +/- 1.1), R+FK (3 +/- 1.1), and CsA-C (2.7 +/- 1.2) than in group C (0.8 +/- 1.2). Numbers of morphologically intact anterior horn cells were higher in groups FK (31.3 +/- 9.9), R+FK (23.2 +/- 4.5), and CsA-C (18.3 +/- 6.8) than in group C (6.3 +/- 4.3). FK506 and chronic administration of cyclosporine A, but not rapamycin, protect the spinal cord from transient ischemia. Although these results are compatible with inhibition of calcineurin in the mechanism of neuroprotective action of these drugs, other effects through different pathways cannot be excluded before further study.

DOES SONIC HEDGEHOG SIGNALING PLAY A ROLE IN ACUTE INFLAMMATORY, ALLOIMMUNE RESPONSES AND CHRONIC ALLOGRAFT REJECTION?

A281 Aims: There is emerging evidence that sonic hedgehog (Shh) pathway contributes to the regulation of peripheral immunity and is proved to be an indirect angiogenic factor regulating expression of multiple angiogenic cytokines and indicate that Shh might have potential therapeutic use for ischemic disorders. We are encouraged by these facts to investigate the role of Shh in acute inflammatory, acute and chronic allograft rejection in our rat models of orthotopic small bowel transplantation. Methods: Orthotopic SBT between DA to LEW strain combination was used. Acute rejection occurs between 5-7 days, but this can be controlled with a unique FK506 pretreatment regimen with a proportion of recipients undergoing chronic rejection. Syngeneic transplant was used as a model of acute inflammatory and controls for acute and chronic allograft rejection. Immunohistochemistry was performed to study the expression on the graft isolated at 7 and 90 days post-transplantation. Results: Patched (Ptc) is expressed in the infiltrated macrophage in the mesentery. Shh, on the other hand is mainly expressed in the small blood vessels and to a less extent in the medium-large the macrophages in the mesentery. The expression pattern of Shh and its receptor Ptc is similar in the different experimental groups. The order of intensity follows: Acute allograft rejection→acute inflammatory →chronirc allograft rejection→long-term survived syngeneic graft. Conclusions: The activation of Shh pathway is remarkable in acute phase of inflammatory responses and enhanced by alloantigen activation. The role of Shh signaling in acute inflammatory and allo-immune responses needs to be further validated using in vitro system. The activation of Shh pathway decreases in long-term syngeneic transplants but retained in CR. Furthermore, Shh signaling expression is found in the small to a less extent in medium and large vessels, it is speculated that Shh may not be involved in allograft vasculopathy in chronic rejection.

Role of FKBP12.6 in hypoxia- and norepinephrine-induced Ca 2+ release and contraction in pulmonary artery myocytes

The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca 2+ release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP 3 receptors (IP 3Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca 2+] i and Ca 2+-activated Cl − and K + currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca 2+] i. FK506-induced [Ca 2+] i increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP 3R antagonist heparin. Hypoxic Ca 2+ response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca 2+] i, but did not alter caffeine-induced Ca 2+ release (SR Ca 2+ content) in PASMCs. Norepinephrine-induced Ca 2+ release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca 2+ and contractile responses by regulating the activity of RyRs in PASMCs.

Rat liver ischemia–reperfusion-induced apoptosis and necrosis are decreased by FK506 pretreatment

The aim of this study was to demonstrate that tacrolimus (FK506) has a hepatoprotective effect by reducing ischemia–reperfusion-induced apoptosis and necrosis, both of which lead to post-surgical liver dysfunction. An ischemia–reperfusion model and primary cultured rat hepatocytes subjected to hypoxic and reoxygenation phases, mimicking the surgical process, were used. c-Jun N-terminal kinase 1/stress-activated protein kinase 1 (JNK1/SAPK1) activation leads to caspase 3 activation, a trigger of apoptosis. The activation status of JNK1/SAPK1 was evaluated by immunoprecipitation or Western-blotting experiments. Apoptosis was assessed by measuring caspase activation and by TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling) reaction. Necrosis was assessed histologically. Tacrolimus improved the survival rate of rats subjected to ischemia–reperfusion. After FK506 pretreatment, the liver necrosis rate was reduced, and ischemia–reperfusion-induced JNK1/SAPK1 activation and apoptosis were significantly decreased. In hypoxia-reoxygenation-subjected hepatocytes, tacrolimus reduced JNK1/SAPK1 and caspase 3 activation. In the liver, tacrolimus prevented ischemia-reperfusion-induced apoptosis and necrosis.

Protective effect of FK506 (Tacrolimus) in pentylenetetrazol-induced kindling in mice

The repeated administration of otherwise subconvulsant dose of pentylenetetrazol (PTZ) is known to produce chemical kindling in animals. In our study, chronic administration of subconvulsant dose of PTZ (40 mg/kg) produced chemical kindling in mice. Pretreatment with l-arginine (50–100 mg/kg ip) potentiated the PTZ-induced kindling, whereas N ω-nitro- l-arginine methyl ester ( l-NAME) (10–20 mg/kg ip) showed a protective effect. FK506, a potent neuroprotective agent, dose dependently (0.5–1 mg/kg po) decreased the kindling score. When given in combination, l-NAME potentiated the protective effect of lower dose of FK506 (0.5 mg/kg) on PTZ-induced kindling. l-Arginine (50–100 mg/kg) reversed the protective effect of FK506 (1 mg/kg) and l-NAME (20 mg/kg). Biochemical studies showed the potential role of free radical toxicity in the kindled mice, as there was an increased lipid peroxidation as indicated by elevated malondialdehyde (MDA) and nitrite levels and decrease in GSH and superoxide dismutase (SOD) levels. FK506 pretreatment significantly reversed the elevated MDA and nitrite levels, GSH and SOD depletion induced by PTZ treatment. In conclusion, the results of the present study suggest the possible neuroprotective action of FK506 against PTZ-induced kindling.

Pretreatment with FK506 up-regulates insulin receptors in regenerating rat liver

This report examines the effect of FK506 pretreatment on liver insulin receptor expression in partially (70%) hepatectomized rats. FK506 pretreatment led to an increased insulin receptor number 24 hours after hepatectomy, detected by means of insulin binding and cross-linking procedures. This increase was related to enhanced insulin receptor expression determined by in vitro mRNA translation and Western blot techniques. We also tested the functionality of the expressed insulin receptors by [ 3H] thymidine incorporation into DNA in insulin-stimulated hepatocytes. The results show that FK506 pretreatment elicits an increase in the amount of insulin receptor α–subunits as measured by Western blot. Maximum α-subunit expression recorded 24 hours after surgery was preceded by increased insulin receptor mRNA levels, which were detected 6 hours after hepatectomy. Moreover, in FK506–pretreated rat hepatocytes, obtained from remnant livers 24 hours after partial hepatectomy (PH), the increase in insulin receptor number was associated with improved sensitivity to the hormone. However, in both experimental groups (FK506-pretreated and nonpretreated rats), the sensitivity of hepatocytes toward epidermal growth factor (EGF) showed no significant change, which suggests a specific effect of FK506 on insulin receptor expression. In conclusion, our findings suggest that FK506 pretreatment induces insulin receptor expression in regenerating rat liver and promotes liver regeneration in hepatectomized rats. (H EPATOLOGY 2002;36:555-561.)

Recipient FK506 pretreatment regimens in rat small bowel transplantation: Allograft survival, function, and systemic infection

Purpose: Successful small bowel transplantation requires effective immunosuppression that preserves intestinal function but avoids opportunistic infection. This study aims to evaluate FK506 as a single immunosuppressant in different pretreatment regimens in a rat high responder strain combination. Methods: Lewis → DA rat heterotopic small bowel transplantation was performed. Studied groups were (1) untreated control, n = 12; (2) FK-1, n = 8; (3) FK-3, n = 8. FK506 (2 mg/kg/d, intramuscularly) was given to the recipients for 1 day (FK-1) and 3 days (FK-3) before small bowel transplantation, followed by 2 weeks of subtherapeutic treatment (0.3 mg/kg/d, intramuscularly) after small bowel transplantation. Syngeneic small bowel transplantation also was performed (n = 8). FK blood levels, maltose absorption test, histology, and bacteriology were performed at different postoperative days. Results: Allograft survival was prolonged significantly with FK pretreatment, being more so in FK-3 group (FK-1, 22.2 ± 1.5 d; FK-3, 40.7 ± 14.1 d; control, 6.6 ± 0.8 d; P <.01). In the first postoperative week, FK blood level was significantly higher in FK-3 group (19.8 ± 1.5 ng/mL) than in FK-1 group (5.0 ± 0.4 ng/mL; P <.05). There was no evidence of systemic infection in either FK-treated group. For maltose absorption, control allograft was abnormal on day 7 correlating to severely damaged intestinal architecture. In contrast, FK-treated allografts showed well-protected intestinal structure and normal absorption on days 7 and 21. Conclusion: High FK506 blood levels in the first postoperative week, achieved with FK pretreatment, prolonged intestinal allograft survival and preserved intestinal structure and function without allowing systemic infection. J Pediatr Surg 35:1600-1605. Copyright © 2000 by W.B. Saunders Company.

STAT 6 up-regulation by FK506 in the presence of interleukin-4.

FK506 perturbs normal phosphorylation by inhibition of the PP2B protein phosphatase, calcineurin. Calcineurin activity is required for intracellular signal transduction via the T cell receptor that in turn leads to either TH1, or TH2, -type responses to antigen. This choice of response involves differential phosphorylation of STATS (Signal Transducers and Activators of Transcription) for induction of STAT activity. Interferon-gamma activates STAT1, a TH1-type mediator, and interleukin-4 activates STAT6, a TH2-type mediator. We ask if FK506 biases STAT activation toward a TH2-type response. Cells of the RAW 264.7 mouse macrophage line were treated with interleukin-4, or interferon-gamma, plus or minus FK506, and any effect on STAT6 and STAT1 was compared. Interleukin-4 specifically induced activation of STAT6, and pretreatment with FK506 enhanced this activity. Interferon-y induced STAT1 activity but this was not influenced by FK506 pretreatment. FK506 protects against allograft rejection by inhibiting interleukin-2 production. Such protection may be enhanced by FK506-mediated up-regulation of STAT6 activity.

FK506 with Portal Decompression Exerts Beneficial Effects following Extended Hepatectomy in Dogs

The present study was designed to elucidate the effectiveness of portal decompression and FK506 (FK) pretreatment in extended hepatectomy in dogs. In the first set of experiment the effect of portal decompression was evaluated in two groups of dogs which underwent extended hepatectomies (80%) with or without (control) a side-to-side portacaval shunt. The presence of the shunt significantly (p < 0.05) improved the 7-day survival of the animals (57.1%) when compared with those of the control group (28.6%) and eventually the portal pressure was significantly lower and mean arterial pressure was significantly higher in the shunt group (p < 0.05). Moreover, the animals with lower portal pressure (≤220 mm of saline) had a significantly improved 7-day survival rate than those with higher portal pressure (p < 0.001). To evaluate the role of FK pretreatment in extended hepatectomy, a more severe model of 90% hepatectomy was used in four different groups: portacaval shunt, shunt and FK pretreatment, FK pretreatment, and hepatectomy only (control). Although the shunt improved the survival rate in the 80% hepatectomy model, neither shunt (8.3%) nor FK pretreatment (0%) independently improved the survival of the animals when the hepatectomy was extended to 90%. FK pretreatment significantly improved the survival (33.3%, p < 0.05) and hepatic functions of the animals only in the presence of a shunt. Also hepatic microcirculation measured with a laser Doppler flowmeter was significantly better in the remnant liver of all treated groups than in the control group (p < 0.01). In conclusion, extended hepatectomy (90%) could be performed in otherwise normal liver after FK pretreatment and a side-to-side portacaval shunt, which improve the regenerative response in a stable hemodynamic animal.

Involvement of neutrophils in ischemic injury. I. Biochemical and histopathological investigation of the effect of FK506 on dorsal skin flaps in rats.

The purpose of this study was to investigate the role of neutrophils in ischemic tissue injury and the possible inhibition by pretreatment with FK506, a neutrophilic modulating agent. A dorsal caudally based skin flap (3 x 9 cm) was used as an ischemic injury model in experimental groups. Prior to flap elevation, FK506 at doses of 0.3 mg per kilogram (group 2), 0.5 mg per kilogram (group 3), and 1.0 mg per kilogram (group 4) was given for 3 days intramuscularly. The relationship among neutrophil accumulation (histopathologically), myeloperoxidase (MPO) activity, malondialdehyde (MDA) content (biochemically) of the flap tissue, and flap survival were studied. Skin flaps showed reduced necrosis in the FK506-treated groups (p < 0.08, p < 0.0001, and p < 0.0001 respectively). The increase in accumulation of neutrophils, and MDA and MPO levels (which were induced by ischemia) observed 1 and 24 hours after flap elevation was diminished by FK506 pretreatment. The increased neutrophilic infiltration, and raised tissue MDA content and MPO activity revealed involvement of both free radical production and neutrophils in ischemia. This injury was decreased by FK506, probably by inhibition of neutrophilic chemotaxis, infiltration, and releasing factors.

FK506 Maintains Cellular Calcium Homeostasis in Ischemia–Reperfusion Injury of the Canine Liver

Increased morbidity and mortality following transplantation surgery due to the primary nonfunction and dysfunction of the liver poses a great challenge and has increased the crescendo of research work in this field. In this study, we have tried to address the issue concerning the changes in Ca2+homeostasis and hepatic microcirculation in 90 min of ischemia followed by reperfusion of the liver after FK506 pretreatment. Twenty dogs divided into two groups; group I (0.15 mg/kg/day FK506 for 3 days, im) and group II (control) were used for the measurement of mitochondrial (mit) and total cellular Ca2+by atomic absorption spectrophotometer and hepatic microcirculation with laser Doppler flowmeter. Serum AST leakage was significantly (P< 0.05) suppressed in group I at 6 hr after reperfusion. The percentage gain in mit Ca2+in group I was significantly (P< 0.05) inhibited compared to that in group II at 15 min after reperfusion and also when compared with the preischemic value it was significantly (P< 0.05) elevated at 30 min after reperfusion in group II only. FK pretreatment significantly (P< 0.05) inhibited the overload in total cellular Ca2+at 15 and 30 min after reperfusion. Moreover, hepatic microcirculation was significantly (P< 0.001) better in group I between 2 and 6 hr after reperfusion. In conclusion, the ameliorating property of FK in ischemia–reperfusion may be explained by prevention of the cellular Ca2+overload during the perireperfusion period.

FK506 inhibits renal glomerular thrombosis induced in rats by nephrotoxic serum and lipopolysaccharide

We investigated the effect of the potent immunosuppressive agent, FK506, on experimental glomerular thrombosis in rats by combined injections of nephrotoxic serum (NTS) and lipopolysaccharide (LPS). Either FK506 or placebo was administered intramuscularly three hours prior to injection of NTS that was followed one hour later by LPS. Rats were killed five hours after the LPS injection. Compared with placebo, FK506 pretreatment significantly reduced thrombosis formation, in a dose-dependent manner. FK506 also reduced proteinuria and the rise of serum creatinine level. Early infiltration of polymorphonuclear leukocytes into the glomeruli after LPS injection was significantly suppressed in the FK506 group compared with the placebo group. We also measured serum tumor necrosis factor (TNF) activity by using an L929 fibroblast cytotoxicity assay. Peak serum TNF activity was observed one hour after LPS injection, and FK506 significantly suppressed the elevation. Thrombosis was also developed in athymic nude rats, suggesting thrombosis formation is T cell independent. These data suggest that the FK506 has inhibitory effects on non-lymphocytes and possesses an anti-inflammatory effect in vivo.