Tacrolimus对糖尿病大鼠肾脏足细胞损伤的保护作用及机制

目的探讨Tacrolimus（FK506）是否通过足细胞的保护作用减轻糖尿病大鼠尿白蛋白排泄。方法将40只大鼠按随机数表法分为对照组（C组）、糖尿病（diabetesmellitus,DM）模型组（DM组）、DM＋FKS060．5mg·kg^-1·d^-1给药组（FK5060．5组）及DM＋FK5061．0mg·kg^-1·d^-1给药组（FK5061．0组）,每组10只。采用链脲佐菌素（streptozotocin,STZ）腹腔注射建立糖尿病模型,FK506灌胃给药。4周后大鼠24h尿白蛋白测定采用酶联免疫方法,电镜下观察肾小球足细胞病理组织学改变,应用免疫荧光与Westernblot检测肾组织Nephrin和Podocin表达。结果DM组大鼠24h尿白蛋白排泄率（albuminexcretionrate,AER）明显高于对照组（P〈0．01）,FK5060．5与1．0mg／kg给药组大鼠AER水平明显低于模型组（P〈0．05,P〈0．01）。透射电镜观察DM组肾小球基底膜增厚、结构模糊不清,系膜基质增多,足细胞损伤,与DM组比较,FK5060．5、1．0组肾组织超微结构改变有不同程度改善。免疫荧光显示Nephrin和Podocin在C组大鼠肾小球呈线状均匀分布;DM组大鼠肾小球表达明显减少,且呈颗粒状不均匀分布;FK5060．5组和FK5061．0组Nephrin和Podocin表达不同程度增加,呈线状及颗粒状分布。Westernblot显示DM组Nephrin和Podocin较C组表达明显下降;FK5060．5组和FK5061．0组Nephrin和Podocin量较DM组明显增加（P〈0．01）。结论FK506能减少糖尿病大鼠尿白蛋白排泄,改善肾小球足细胞病变,其机制可能与上调Nephrin和Podocin表达有关。     

Changes in the mononuclear cell subpopulations of rat cardiac transplant recipients administered FK506 for the treatment of ongoing rejection

The inhibitory effect on ongoing rejection and the changes that occurred in mononuclear cell subpopulations were compared between four groups of rats treated with FK506 or steroids. Group 1 was given no immunosuppressive drugs, group 2 was given FK506 from the day of grafting, group 3 was commenced on FK506 on the 4th day after grafting, and group 4 was commenced on methylprednisolone (MP) on the 4th day after grafting. The garft survival times in groups 2 and 3 were significantly longer than those in groups 1 and 4, and there were fewer CD3⁺ and CD4⁺ T lymphocytes in the peripheral blood in the groups treated with immunosuppressive drugs than in group 1. In group 4, the levels in both the peripheral blood and thymus were significantly lower than those in the groups treated with FK506 despite the fact that graft rejection occurred soon after the discontinuation of steroid administration. Moreover, the levels of interleukin-2 receptors and macrophages in groups 2, 3, and 4 were significantly lower than that in group 1 postoperatively; however, the number of macrophages in groups 2 and 3 was significantly lower than that in group 4 on the 10th day after transplantation. The findings of this study demonstrated that FK506, even if administered after rejection has begun, might inhibit the subsequent extensive allograft rejection more specifically and effectively than steroids, and that the measurement of a marker for macrophages in the peripheral blood could be useful for the detection of rejection following allograft transplantation in rats.     

Comparison of ELISA method versus MEIA method for daily practice in the therapeutic monitoring of tacrolimus.

BACKGROUND: For the adequate management of transplant patients on tacrolimus therapy, it is important to obtain optimal blood concentrations. The purpose of this study was to determine the most appropriate method for daily practice of tacrolimus determination in whole blood. We compared enzyme-linked immunosorbent assay (ELISA) with microparticle enzyme immunoassay (MEIA), using European controls and blood samples from organ graft recipients treated with tacrolimus. Time, practicability and cost were considered also. METHODS: The assays were performed according to the procedures detailed in the product inserts. In five European controls and 40 blood samples from kidney and liver transplant patients, we determined the blood levels of tacrolimus by both MEIA and ELISA tests. RESULTS: MEIA gave more reliable results with the European controls (y=1.078x+0.092; r=0.996) than ELISA (y=0.956x+1.307; r=0.946). For the patient samples, the correlation between the two tests was 0.85 and the extreme range of values was +65% and -56% for ELISA vs MEIA. Although the manufacturer of the ELISA test used claims the best sensitivity and precision, in our experience the MEIA test was quicker and cheaper. CONCLUSIONS: MEIA provides a quick, reliable and easy-to-handle method for routine monitoring of tacrolimus blood levels.     

口服他克莫司血药浓度-时间曲线下面积

目的　探讨口服他克莫司 (Tacrolimus ,FK5 0 6 )的药代动力学规律 ,寻求临床上准确反映早期药物浓度时间曲线下面积 (AUC)的监测方法。　方法　 16例肾移植受者首剂口服 0 .0 75mg/kgFK5 0 6后 ,采用ELISA法测定服药后 0 .5、1.0、1.5、2 .0、3.0、5 .0、8.0、12 .0h时间点血药浓度 ,采用 3p87药代动力学计算程序将测得的FK5 0 6浓度自动拟合计算出AUC ,并分别将各时间点血药浓度与AUC进行相关性检验 ,计算相关系数。　结果　AUC变化范围为 4 4 .4 0～ 15 8.0 1μg·h-1·L-1,平均 ( 92 .2 3± 34.97) μg·h-1·L-1,个体间AUC可相差 4倍 ;血药谷浓度Cmin与AUC之间的相关性有显著差异 (P <0 .0 0 1,rmin=0 .6 5 0 )。　结论　首剂口服同一剂量FK5 0 6后 ,个体间药物浓度时间曲线下面积差异很大。Cmin能准确反映首剂口服FK5 0 6后的AUC ,临床上可通过监测Cmin达到FK5 0 6早期剂量的个体化     

The superior effect of the combination of FK 506 and deoxyspergualin on rat cardiac allograft survival

In this present study, the effects of FK 506 and 15-deoxyspergualin (DSG), with respect to dose, timing, and combination, were investigated in an ACI-to-LEW rat cardiac allograft model. FK 506 was adminstered intramuscularly for 14 days starting on day 0 after grafting, while DSG was given intraperitoneally for 7 days starting on day 0,4, or 7 after transplantation. FK 506 or DSG monotherapy prolonged cardiac allograft survival in dose-dependent manners, and the minimum effective dose for overcoming rejection was 0.1 mg/kg per day in the case of FK 506 and 1.0 mg/kg per day for DSG. The graft survival rate was higher with administration of DSG starting on day 4 on day 0 after transplantation. A low dosage of FK 506 strating on day 0, in combination with DSG starting on day 0 or day 4 (but not on day 7), had a synergistic effect in prolonging allograft survival for 14.0±3.3 days and 25.4±8.2 days, respectively. The most effective combination treatment schedule for prolongation of allograft survival was FK 506 starting on day 0 and DSG starting on day 4 after transplantation.     

Bioavailability and costs of once‐daily and twice‐daily tacrolimus formulations in de novo kidney transplantation

The use of once‐daily tacrolimus in de novo kidney transplantation is increasingly common. Therefore, we were interested in bioavailability aspects of novel once‐daily tacrolimus (LCPT, Envarsus) and once‐daily tacrolimus extended‐release formulation (ER‐Tac, Advagraf) compared with twice‐daily immediate‐release tacrolimus (IR‐Tac, Prograf). Furthermore, we calculated the costs. Kidney allograft recipients on tacrolimus‐based immunosuppression within 2 clinical trials were included in a single‐center analysis. The tacrolimus formulations were compared with respect to daily doses, doses per body weight, trough levels, and concentration‐dose (C/D) ratio over 12 months. Intrapatient variability in trough levels and C/D ratios after 3 months was calculated. For the calculation of tacrolimus costs, German list prices were used. Eighty patients (21 with LCPT, 23 with IR‐Tac, and 36 with ER‐Tac) were analyzed. Pharmacokinetic comparisons revealed significantly higher bioavailability of LCPT at all visits. The variability of trough levels and C/D ratios in general was high and highest in LCPT patients. Different dose requirements translated into different costs. Median treatment costs during the first year were 7.825€ (IQR 6.195‐8.892€) for LCPT, 9.813€ (IQR 7.630‐16.832€) for IR‐Tac, and 9.838€ (IQR 7.503‐ 13.541€) for ER‐Tac (Kruskal‐Wallis test, P = .003). The 3 tacrolimus formulations exhibit different dose requirements, exposure, and costs in favor of LCPT.     

他克莫司和环孢素A对大鼠肾脏转化生长因子及其受体表达影响的比较

目的比较他克莫司（FK506）和环孢素A（CsA）所致慢性肾毒性大鼠模型肾组织转化生长因子目（TGF-1β）及其受体（TβRⅡ）的表达。方法分别以FK506和CsA灌胃复制大鼠FK506和CsA慢性肾毒性模型，观察大鼠的一般情况，计算肌酐清除率，观察大鼠肾组织病理变化，以免疫组织化学法检测肾组织中TGF-1β、TβR Ⅰ、TBRⅡ蛋白表达的变化，原位杂交法检测肾组织中TβR Ⅰ mRNA及TβR Ⅱ mRNA表达的变化。结果CsA组和FK506组的肾小管、小管间质和人球小动脉均有损伤，但CsA组的损伤明显较FK506组重（P〈0．05）。正常对照组大鼠肾组织中仅见少量TGF-[3，、TβR I和T13RⅡ表达，CsA组和FK506组的TGF-β，、TβR I和TβR Ⅱ表达均明显增加，但FK506组稍轻；正常对照组大鼠肾组织中仅见少量TβR I mRNA、TβR Ⅱ mRNA表达，CsA组和FK506组TβR I mRNA、TβR Ⅱ mRNA明显表达，但FK506组较CsA组为轻。结论FK506的慢性肾毒性弱于CsA，它所诱导的大鼠肾组织中TGF-β，及其受体TβR I和TβR Ⅱ的表达均低于CsA。     

他克莫司和雷帕霉素对肝癌肝移植患者Foxp3+ Treg的影响及防治排斥反应的研究

目的 探讨免疫抑制剂他克莫司和雷帕霉素对肝癌肝移植受者Foxp3+ Treg产生的影响及其防治排斥反应的疗效.方法 自移植后第2个月到第12个月,每月采血,采用实时荧光定量PCR法检测他克莫司组和雷帕霉素组肝癌肝移植受者新鲜外周血单个核细胞中Foxp3 mRNA的表达水平,通过同期术后观察和实验室检查,比较两组受者间Foxp3 mRNA表达水平和急性排斥反应发生率的差异.结果 他克莫司组受者的外周血单个核细胞中Foxp3 mRNA表达水平(0.1032±0.0943)明显低于雷帕霉素组受者(1.2136±0.6738),差异有统计学意义(t=5.1610,P＜0.01);雷帕霉素组患者比同期他克莫司组受者术后急性排斥反应发生率明显减低,差异有统计学意义(x2=2.2222,P＜0.05).结论 他克莫司抑制了肝癌肝移植术后免疫耐受的诱导,而雷帕霉素可能参与了免疫耐受的诱导和维持;雷帕霉素对肝癌肝移植受者防治排斥反应的效果更好.     

Efficacy of transient treatment with FK506 in the early phase on cyclophosphamide-induced bone marrow chimerism and transplant tolerance across MHC barriers

BACKGROUND: The use of mixed allogeneic bone marrow chimerism to induce donor-specific transplantation tolerance has been extensively demonstrated. In the present study, we assessed the effect of combined use of a short course of FK506 and a single-dose cyclophosphamide (CYP) on the induction of tolerance and development of GVHD after allogeneic BMT. MATERIALS AND METHODS: Lewis rat (RT1(l)) recipients received BMT from Brown Norway (RT1(n)) donors on the next day after injection of CYP at a dose of 200 mg/kg. The recipients were further treated with no FK506 (n = 8), 0.3 mg/kg/day FK506 on days 10-16 (n = 6), or the same dose of FK506 on days 0-6 (n = 6). In a subgroup of animals, heterotopic heart transplantation was performed to investigate transplantation tolerance. RESULTS: Six of eight recipient rats that did not receive FK506 died of severe GVHD, while high levels of chimerism were induced. Recipients of FK506 in the later phase developed mild transient GVHD around 2 to 3 weeks after BMT and recovered thereafter; however, the level of chimerism was significantly decreased (2.8 +/- 2.3% on day 100). Treatment with FK506 in the early phase completely prevented the development of GVHD and induced stable allogeneic chimerism in the long-term (13.8 +/- 8.3% on day 100). These recipients with stable chimerism accepted subsequent BN heart allografts indefinitely (>200 days x 5), while rejecting third-party (BUF) heart allografts by day 12. CONCLUSIONS: Early transient FK506 promotes the induction of stable bone marrow chimerism without GVHD after BMT with CYP pretreatment. The timing of treatment with FK506 is critical with a view to preventing GVHD and inducing stable long-lasting chimerism.     

Two-year follow-up study of the efficacy and safety of FK 506 in kidney transplant patients

Abstract We conducted a 2-year follow-up study of the efficacy and safety of FK 506 in 104 kidney transplant patients at 32 sites in Japan. The initial daily oral dose of FK 506 was 0.3 mg/kg, which was gradually reduced to 0.15 mg/kg by month 10 and remained stable thereafter. The mean trough level of FK 506 in whole blood and the mean serum creatinine level in year 2 were 7.9 ng/ml and 1.9 mg/dl, respectively. Patient and graft survival rates for all patients were 97% and 92%, respectively. Forty-ix patients (44%) experienced rejection episodes, and 84% of these episodes occurred within 3 months after transplantation. The principal adverse reactions to FK 506 therapy were hyperglycaemia, renal dysfunction and hyperkalaemia. Most of these events were dose-dependent, and disappeared or ameliorated following reduction of the FK 506 dose.     

Influence of an orally administered calcium-binding cation exchanger on calcium metabolism in the rat

Summary The kinetics of calcium metabolism in the rat were analysed by doing a calcium balance and an assay with ⁴⁵Ca after feeding a calciumbinding ion exchanger during 3 weeks. Serum and urine concentrations of inorganic phosphate and of calcium were estimated. The increase in the size of the exchangeable calcium pool, the activation of bone turnover, the hypophosphataemia and hyperphosphaturia, as well as hypercalciuria are evidence for a reactive hyperparathyroidism. The results of these experiments call for further investigations concerning the influence of cation exchangers on bone metabolism under clinical conditions.     

The effect of FK506 on Warm Ischemia and reperfusion Injury in the Rat Liver

The protective effect of FK506 on hepatocytes against ischemia and reperfusion injury was examined by evaluating the following: the high energy phosphorus metabolism obtained using 31P magnetic resonance spectroscopy (³¹P-MRS) and the tissue blood flow of the liver in ischemia and the reperfusion process, mitochondrial glutamic oxaloacetic transaminase (m-GOT) and glutamic pyruvic transaminase (GPT), the survival rates of the animals, a histological study and immunohistological staining for intercellular adhesion molecule-1 (ICAM-1) in the liver after ischemia. The rats were treated with FK506 1 mg/kg/day i.m. for 4 days before testing. Ischemia was induced by clamping the hepatoduodenal ligament for 30 min. In³¹P-MRS, the recovery of the hepatic energy status after ischemia, evaluated by -ATP/inorganic phosphate (Pi), was significantly better in the FK506 group. It also coincided with the recovery of tissue blood flow monitored with a laser Doppler flowmeter. In the histological examination, the congestion observed in the periportal region of the control group was mild, while there was less induction of ICAM-1 in the endothelial cells of the portal veins and hepatic veins in the FK506 group. From these findings, we concluded that FK506 had a protective effect on hepatocytes against warm ischemia and reperfusion injury, and the mechanism for this could partially be attributed to improved tissue blood flow after ischemia by the modulation of immunological events.     

Corticosteroids and concomitant medication in the European multicentre study of FK 506 and cyclosporin in primary liver transplantation

Abstract The steroid-paring effect and the use of concomitant medication during the treatment of liver transplant patients with the novel immunosuppressant FK 506 were evaluated within the European multicentre, randomized, parallel-group study in liver transplantation. Patients undergoing primary liver transplantation were randomized to treatment with FK 506 ( n = 267) or with a cyclosporin-based immunosuppressive regimen ( n = 273). The total cumulative steroid usage was significantly reduced in the FK 506 treatment group, which is likely to have resulted from the lower incidence of acute rejection in these patients. The number of patients receiving antidiabetic, diuretic and antihypertensive therapy did not differ between the two treatment groups, even though the incidence of diabetes mellitus and oliguria was significantly higher in the FK 506 group. It can, therefore, be assumed that in a number of such cases the severity of these events was very mild necessitating no specific therapy.     

Immunosuppressive effect of the gut microbiome altered by high‐dose tacrolimus in mice

The alterations induced in gut microbiota by tacrolimus may affect immune function and organ transplantation. Mice were treated with high‐dose tacrolimus for 14 days. The fecal microbiota were analyzed by pyrosequencing the 16S rRNA genes, and the effect on metabolism was predicted using the sequence data. The subgroups of T cells in the serum, gut‐associated lymphoid tissue, and draining lymph nodes were determined by flow cytometry. Tacrolimus treatment significantly altered the relative abundance of Allobaculum, Bacteroides, and Lactobacillus and CD4⁺CD25^hiFoxP3⁺ regulatory T cells in the colonic mucosa and the circulation. These were significantly increased after either tacrolimus treatment or treatment by fecal microbiota transfer from tacrolimus‐treated donors. Further, treatment with low‐dose tacrolimus plus fecal microbiota transfer from high‐dose tacrolimus–altered mice increased skin allograft survival rate in a skin transplantation model. Thus, high‐dose tacrolimus alters the compositions and taxa of the gut microbiota. Administration of these conditioned gut microbiota plus low‐dose tacrolimus resulted in regulation of colonic and systemic immune responses and an increased allograft survival rate. This study demonstrated a new strategy for controlling allograft rejection by combining an immunosuppressive agent with gut microbiome transplantation.     

New morphological changes induced by FK506 in a short period in the rat kidney and the effect of superoxide dismutase and OKY-046 on THEM: the relationship of FK506 nephrotoxicity to lipid peroxidation and change in production of thromboxane A2 in the kidney

Juxtaglomerular (JG) hyperplasia and tubular damage along with a decrease in the urine creatinine level induced by FK506 in rat kidney have already been reported in previous paper by us [6]. In this paper, we document the relationship of FK506 nephrotoxicity to the change in the production of thromboxane (Tx) A₂ and the lipid peroxidation of the cellular membrane in the rat kidney in order to clarify its morphogenesis. The urinary excretion of TxB₂ increased with FK506 administration even on day 1 (P < 0.02). Histologically, OKY-046 (thromboxane synthetase inhibitor) decreased tubular damage, although JG hyperplasia was not eradicated, while biochemically the excretion of TxB₂ decreased significantly (P < 0.02), and both the decrease in the urine creatinine level and the increase in the N-acetyl-β,d -glucosaminidase (NAG) index were relatively smaller. Although the FK506-induced morphological and biochemical changes could not be prevented by the continuous administration of superoxide dismutase (SOD) 30000 U/kg daily, the malondialdehyde content in renal tissue removed 1 h after FK506 administration had increased. These data suggest that FK506 nephrotoxicity is related to the change in the production of TxA₂ and lipid peroxidation of the cellular membrane. However, other mechanisms such as the involvement of sympathomimetic effects of FK506 and other vasoconstrictive factors cannot be rules out.     

他克莫司对犬急性脊髓损伤神经保护作用的实验研究

目的探讨他克莫司（FK506）对犬急性脊髓损伤的神经保护作用。方法采用Allen＇s法制成犬急性脊髓损伤实验模型。动物随机分为：A组（n=8）,经动脉插管注入生理盐水;B组（n=8）,FK5060.18mg／kg;C组（n=8）．FK5060.3mg／kg。B组、C组均在致伤后2h经动脉插管单次给药。致伤后行脊髓MRI影像学检查、脊髓功能评分、脊髓组织病理观察、神经丝蛋白（NF200）及胶质纤维酸性蛋白（GFAP）的免疫组织化学分析。结果脊髓功能评分C组优于A组（P〈0.05）;B组优于A组,但无统计学差异;MRI显示：脊髓损伤后C组病变范围小,恢复快,B组次之．A组最差。C组NF和GFAP表达高于A组（P〈0.05）,B组与A组无统计学差异。结论局部应用FK506（0.3mg／kg）对急性脊髓损伤具有神经保护作用,可减轻继发损伤,加快脊髓功能的恢复;局部应用FK506对急性脊髓损伤治疗有一定的剂量-效应依赖关系。     

Toxicology of FK506 in the cynomolgus monkey: a clinical,biochemical, and histopathological study

We investigated clinical, biochemical, and histopathological parameters in FK506-treated cynomolgus monkeys. Eight monkeys given oral FK506, 1 (n = 4) or 10 (n = 4) mg/kg daily, survived the 90 days of treatment apparently in good health and without significant changes in biochemical and histopathological parameters, as did 2 control monkeys except one monkey on 10 mg/kg/day FK506 orally, who was found to have a malignant lymphoma. In contrast, monkeys given intramuscular FK506 1 mg/kg daily (n = 4) had to be sacrificed at day 20, 25, 32, and 47 because of severe illness. They showed abnormal biochemical parameters (increased serum urea and aspartate aminotransferase activity) and major histopathological changes in the kidney (mesangial cell proliferation and acute tubular necrosis), pancreas (depletion of beta cells), liver (steatosis), and heart (cardiomyopathy). Intramuscular administration of 1 mg/kg daily resulted in serum levels ranging from 10 to 15 ng/ml, while oral administration at a dose of 1 or 10 mg/kg daily resulted in equal or even higher serum levels (range 2–70 ng/ml). Thus, the height of the serum trough level of FK506 using the enzyme immunoassay is not related to the toxicity of FK506 in cynomolgus monkeys.     

FK 506 and cyclosporin each block antigen-induced T cell receptor signalling that is dependent on CD 4 co-receptor and operates in the absence of detectable cytoplasmic calcium fluxes

Abstract The T cell hybridoma "171", which lacks CD4 but expresses T cell receptor (TCR) for hen egg white lysozyme, requires introduction of wild-type CD 4 for antigen-mediated induction and secretion of interleukin-2 (IL-2). Mutant CD 4, which fails to associate with the tyrosine kinase p561ck does not support IL-2 secretion, suggesting that a role of CD 4 is to bring cytoplasmic p561ck into alignment for signal transduction to the IL-2 promotor. Using 171, 171-CD4 (wild-type) and 171-CD4 (mutant), we found that IL-2 secretion was inhibited by FK 506 and cyclosporin but not by rapamycin. However, this inhibition was not associated with calcium fluxes since no change in cytoplasmic free calcium levels ([Ca],; resting level 80 n M ) was detectable during antigen stimulation of the 171 or 171-CD4 cells. Thus, although FK506 and cyclosporin inhibited calcium-dependent signalling to the IL-2 promoter via inhibition of the protein phosphatase calcineurin, it is possible that IL-2 induction via TCR/CD4 requires an FK.506 (and cyclosporin) sensitive step which is independent of cytoplasmic calcium changes.