Abstract
BACKGROUND: The introduction of cyclosporin A (CsA) has greatly enhanced the early survival rate of kidney graft, but the long-term graft survival rate is still limited. Whether tacrolimus prevents chronic allograft nephropathy (CAN) and prolongs survival time is now becoming a hot spot in field of renal transplantation.

OBJECTIVE: To investigate the feasibility and safety of converting CsA to tacrolimus (FK506) in preventing progression of CAN.

DESIGN: Observation and controlled trial.

SETTING: Department of Urological Organ Transplantation, Center of Organ Transplantation, the Second Xiangya Hospital, Central South University.
PARTICIPANTS: A total of 73 patients who had received kidney transplantation at the Department of Urological Organ Transplantation, Center of Organ Transplantation, the Second Xiangya Hospital of Central South University from April 2001 to October 2005, and had been diagnosed as CAN by graft biopsy (42 male patients and 31 female patients; age ranged 19–69 years), were enrolled in the study approved by the ethics committee of this hospital after their written informed consents. CsA soft capsules (Hangzhou Zhongmei Huadong Pharmaceutical Limited Company or Huabei Pharmaceutical Limited Company); mycophenolate mofetil capsules (Shanghai Roche Pharmaceutical Limited Company); prednisone acetate tablets (Second Xiangya Hospital of Central South University); tacrolimus capsules (Fujisawa Pharmaceutical Limited Company).

METHODS: Seventy-three patients voluntarily participated in CsA group (n =30) or FK506 group (n =43). The two groups were homogenous regarding patients’ sex, age and general data (P > 0.05). Patients in the CsA group were continued on their initial immunosuppression protocol, which consisted of CsA, mycophenolate mofetil and prednisone acetate. In the FK506 group, CsA was stopped, and FK506 was started at a dose of 0.08–0.1 mg/(kg·d) 24 hours later, twice daily, administered 2 hours after breakfast and supper. Three days later, the blood trough concentration of FK506 was tested and adjusted to a target range of 5–8μg/L. FK506 dosage adjustment was based on the blood trough concentration, serum creatinine (SCr) and its side effects. All 73 patients were treated for 12 months.

MAIN OUTCOME MEASURES: SCr, glomerular filtration rate (GFR), 24-hour urine protein excretion, serum total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL) and the toxic side effects of calcineurin inhibitors (incidences of tremor, hyperglycemia and hypertension) were monitored during a follow-up of over 12 months.

RESULTS: A total of 73 patients were involved in the result analysis.①12 months after conversion, the level of SCr was statistically reduced and GFR levels were markedly elevated in the FK506 group compared with the CsA group (P < 0.01). TC, TG and LDL levels in the FK506 group were significantly lower than those in the CsA group (P < 0.01).②Compared with the CsA group, the incidence of tremor was obviously increased [30% (9/30), 5% (2/43), P < 0.01] and the incidence of hypertension was obviously decreased [56% (24/43), 83% (25/30), P < 0.05] in the FK506 group.

CONCLUSION: Conversion from CsA to FK506 can postpone renal dysfunction, reduce proteinuria and improve hyperlipidemia. FK506 treatment is an effective therapy in slowing the progression of CAN.

INTRODUCTION

Cyclosporin A (CsA) has remarkably enhanced the early graft survival rate, but the problem of long-term graft dysfunction has not been solved yet[1]. Chronic allograft nephropathy (CAN) is one of the main factors to influence the graft long-term survival rate with its cause and mechanisms unrevealed, and there is not any effective therapy to treat CAN until now[2]. Therefore, we undertook the present study to determine if a conversion from CsA to FK506 in patients with biopsy-proven CAN could ameliorate the progression of renal dysfunction.

SUBJECTS AND METHODS

Subjects
From April 2001 to October 2005, more than 400 kidney transplantations had been finished at the Center of Organ Transplantation in the Second Xiangya Hospital of Central South University. Seventy-three patients who had been diagnosed as CAN by graft biopsy were enrolled in this study with their written informed consents. The primary disease was all chronic glomerulonephritis. They were all first time kidney transplantation with cadaveric donor (all donors signed written informed consents). Each pair of donor and receptor was ABO compatible and Rh positive. The immunosuppression protocol after transplantation was CsA, Mycophenolate Mofetile (MMF) and Prednisone (Pred). Posttransplantational time was 11–53 (mean 27) months. The level of serum creatinine (SCr) was 147.2–324.7 (mean 201.8) μmol/L.

Methods
Diagnostic criterion
Clinical criterion: Posttransplantational time >
6 months, progressive graft dysfunction without any other reasons, SCr > 133μmol/L, always complicated with hypertension, proteinuria and so on. Colored Doppler Flow Imaging hints elevated resistant index in branches of the graft artery.
Pathohistological criterion: Graft biopsy revealed tubular atrophy, extracellular matrix sedimentation, interstitial fibrosis, glomerulosclerosis and obliterative arteriopathy, which were typical pathologic changes in CAN.

Group and treatment
According to converting or not, 73 patients were randomly divided into two groups: ①CsA group (n =30), 17 male and 13 female, the age of transplantation was 21–69 (mean 33.5) years old, dialysis time was 1–15 (mean 8.1) months, the positive rate of complement cytotoxicity (CDC) was below 10%, the positive rate of panel reactive antibodies (PRA) was below 10%, the organ hot ischemic time was 5–13 (mean 7.5) minutes, cold ischemic time was 4–19 (mean 7.8) hours. At the beginning of the study, we maintained on CsA therapy and kept the lower blood CsA valley concentration in the range of 100–150 mg/ml by blood CsA concentration monitoring, all patients received MMF 1.5 g/day as well as Pred 10 mg/day in 6 months’ posttransplantational time and reduced to 5 mg/day after 2 years.②FK506 group (n =43), 25 male and 18 female, the age of transplantation was 19–64 (mean 34.2) years old, dialysis time was 1–13 (mean 7.9) months, the positive rate of CDC was below 10% and the positive rate of PRA was below 10%, the organ hot ischemic time was 5–15 (mean 7.6) minutes, cold ischemic time was 4–22 (mean 8.0) hours. At the beginning of the study, the dosage of MMF and Pred was the same as CsA group. Before conversion, all items aforementioned as well as renal function, 24-hour urine protein excretion, the grades of CAN(Banff 97 Schema) and serum lipids in the two groups had no statistical difference. All patients didn’t take angiotensin converting enzyme inhibitors, angiotensin receptor blockers and any sort of antihyperlipidemic drugs.

Converting scheme
In the FK506 group, CsA was stopped, 24 hours later, FK506 was started at a dose of 0.08–0.1 mg/(kg·d), twice daily. Three days later, the blood trough concentration of FK506 was tested and adjusted to a target level in the range of 5–8 μg/L. FK506 dosage adjustment was based on the following principles: ①When the blood concentration > 10 ng/mL, we reduced 1/4–1/3 of the FK506 dosage and retested the blood concentration one week later.②When SCr kept going up and exceeding its baseline more than 30%, we reduced 1/4–1/3 of the FK506 dosage and retested SCr one week later, if it didn’t stop going up, we reduced FK506 to half of its basic dosage or even stopped using it.③When the patients were complicated with hyperglycemia, we reduced FK506 to half of its dosage.④When the patients were complicated with server infection, we stopped using immunosuppressants for a few days and reassumed the primary immunosuppressive protocol as soon as the infection was brought under control.
Monitoring items
Renal Function: Test blood urea nitrogen, SCr, glomerular filtration rate (GFR) which was calculated according to the Cockcoft-Gault equation).
Quantity of 24-hour urine protein excretion (using Picric Acid Method).
Blood lipids: Fasting 12 hours blood lipids, including serum total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL).
FK506 blood concentration: Using ELISA method to test the concentration, once a week in the first month, twice two weeks in the second month and then once a month.

Statistical analysis
All data were analyzed by SPSS 11.0 software by comparing the CsA group and the FK506 group with unpaired t-test for the continuous variables. Data were expressed as Mean±SD deviation, and P < 0.05 level was set as significant.

RESULTS

Changes of the graft function
After 12 months of follow-up, in the CsA group, the SCr level rose from (198.3±46.7) μmol/L to (245.4±52.8) μmol/L, GFR fell from (48.17±3.02) mL/min/1.73 m2 to (40.58± 2.49) mL/min/1.73 m2; in the FK506 group, the SCr level fell from (209.6±50.4) μmol/L to (194.8±42.5) μmol/L, GFR rose from (48.08±3.15) mL/min/1.73 m2 to (50.14± 3.92) mL/min/1.73 m2, there were statistical differences between the two groups (P < 0.01) (Table 1). In the CsA group, 2 patients lost their graft function and restarted hemodialysis; in the FK506 group, all patients maintained steady renal function.

Changes in 24-hour urine protein excretion
After 12 months of follow-up, in the CsA group, the quantity of 24-hour urine protein excretion rose from (3.2±0.6) g to (3.9±0.7) g; in the FK506 group, the quantity of 24-hour urine protein excretion fell from (3.2±0.8) g to (2.0±0.5) g, there were statistical differences between FK506 group and CsA group (P < 0.01) (Table 1).
Changes in blood lipids
After 12 months of follow-up, in the CsA group, TC, TG, LDL were (6.94±1.37) mmol/L, (3.14±1.38) mmol/L, (3.82±0.89) mmol/L respectively, no significant changes compared to the baseline values. While in the FK506 group, except for HDL, TC, TG, LDL fell to (5.19±0.73) mmol/L, (1.86±0.84) mmol/L, (3.03±0.71) mmol/L respectively, there were statistical differences between the two groups (P < 0.01) (Table 1).

Side effects of drugs
Nine patients complained tremor 2–3 weeks after the administration of FK506, revealed higher incidence than that in CsA group (2 cases) (P < 0.01). Five patients in FK506 group were found slight hyperglycemia, when we reduced the dosage of FK506 and Pred, 4 of them returned to be normal, while one patient needed to use insulin to control hyperglycemia, in CsA group, there were 2 cases of posttransplantational diabetes mellitus (DM), there was no significant difference between the two groups. Twenty-four cases of hypertension were diagnosed in FK506 group while this number in CsA group was 25, FK506 group had a notable lower incidence of hypertension than CsA group (P < 0.05). Five patients complained gum hyperplasia because of CsA administration, when they were converted to FK506 for a few months, their appearances improved. Incidence of gastro-intestinal symptoms such as bloating, diarrhea in the two groups hinted no significant difference. There was no viral infection and no server impaired liver function.

DISCUSSION

The introduction of CsA has greatly improved the short-term survival rate of grafts, but the long-term survival rate is still limited by CAN. CsA has a series of side effects such as nephrotoxicity, hypertension, metabolic disturbances of blood lipids, etc, and it is the main cause of CAN in the triple immunosuppressive regimen[3]. FK506 and CsA all belong to calcineurin inhibitors (CNI), the mechanisms of FK506 are as follows: in the way of combining with endogenous endocellular calcineurin receptor (FKBP), to inhibit the activity of plasmal calcineurin, so as to block the transcription of Interleukin 2 (IL-2), and finally inhibit T lymphocyte activation[4,5], so FK506 can be more efficient in immunosuppression than CsA and it can prevent acute rejection more efficiently. As scientists go deep into the research of FK506, they find that on the basis of enhancing immunosuppression, FK506 can also slow the progression of CAN, the potential mechanisms include:①The immunosuppressive efficacy of FK506 is 50–100 times than that of CsA, it can not only be used as basal immunosuppressants to prevent rejection but also be effective to steroid-resistant and intractable rejection. Thus the administration of FK506 will play an important part in preventing CAN [6]. In this study, in 12 months’ follow-up, there was no acute rejection happened in FK506 group, while 2 cases of acute rejection in CsA group and finally caused graft lost.②The nephrotoxicity of CNI is a very important factor in the progression of CAN, as the dosage of FK506 clinically prescribed is much lower than that of CsA , it will notably lessen the nephrotoxicity of CNI and slow the progression of CAN[3,7].③Hypertension and hyperlipidemia are important risk factors in the occurrence and progression of CAN[8-9]. CsA can activate rennin-angiotensin system and influence the constriction/dilation function of renal vessels to cause hypertension, it can also cause a high level of LDL and enhance the side effects of steroids to cause hypertension. On the contrary, FK506 doesn’t bring these side effects. So after conversion, hypertension and hyperlipidemia will be more efficiently controlled and the graft function will be improved[10-11]. In this study, after 12 months of follow-up, in FK506 group, the level of TC, TG and LDL were (5.19±0.73), (1.86±0.84), and (3.03±0.71) mmol/L respectively, the incidence of hypertension was 55.8% (24/43), they were all at a significantly lower level than that of CsA group (P < 0.01, 0.05).④CsA can injure P-glycoprotein and make it accumulated in the cell, the accumulation will injure proximal tubule and finally cause tubular interstitial fibrosis[12-13]. In vivo and in vitro experiments reveal that CsA stimulate tumor growth factor beta 1 (TGF-β1) to produce and express, which will cause fibrosis and stimulate platelet derived growth factor (PDGF) to produce and finally cause smooth muscle cell hyperplasia, which will play an important role in the progression of the graft arteriosclerosis. Different from CsA, FK506 has the same binding site with TGFβ receptor which is called FKBP-12, it will interfere the expression of TGFβ by competitive combination, and may ameliorate graft fibrosis and finally slow the progression of CAN[14-15]. Proteinuria is one of the important characteristics in the progression of CAN[16], meantime, protein in the urine will sediment in the basement membrane and protein cast will block the tubule, which will accelerate the progression of CAN[17-18]. In FK506 group, after 12 months of conversion, the level of 24-hour urine protein excretion fell from (3.2±0.8) g to (2.0±0.5) g, significantly lower than that of CsA group (P < 0.01), it indicated that FK506 could efficiently improve proteinuria of CAN patients and delay the progression of renal dysfunction.
The side effects and toxicity of FK506 have positive correlation with its dosage. Maintain a target FK506 valley concentration level in the range of 5–8 ng/ml is considered to be safe in a 6-month posttransplantational patient[19-20]. In this study, the incidence of tremor was higher in FK506 group than in CsA group, but when we properly reduced the dosage of FK506, the symptoms of tremor was improved. The incidence of hypertension in FK506 group was obviously lower than that of CsA group (P < 0.05). There was no statistical difference on the incidence of hyperglycemia between the two groups, which indicate that low dosage of FK506 with well controlled blood concentration do not increase the risk of posttransplantational DM.
In the study, the level of SCr in FK506 group fell from (209.6±50.4) μmol/L to (194.8±42.5) μmol/L, and GFR rose from (48.08±3.15) mL/(min/1.73 m2) to (50.14±3.92) mL/ min/1.73 m2, but when we compared between pre- and post-conversion in FK506 group, we found there was no statistical difference, this result indicated that conversion from FK506 to CsA did not reverse the graft’s pathologic change of CAN and could not improve the graft’s function significantly. The conversion can only slowed the progression of renal failure and increase the graft’s long-term survival rate.

REFERENCES

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他克莫司替换环孢素A对慢性移植肾肾病进展的影响☆

谢续标，彭龙开，彭风华，余少杰，王 彧，姜 奕，蓝恭斌，方春华，聂曼华
中南大学湘雅二医院器官移植中心泌外器官移植科，湖南省长沙市 410011
谢续标☆，男，1970年生，湖南省洞口县人，汉族，1993年湖南医科大学毕业，博士，副教授，主要从事肾移植的基础与临床研究。
摘要
背景：环孢素A的应用显著提高了移植肾的早期存活率，但远期移植物失功并未得到明显改善。他克莫司能否延缓慢性移植肾肾病进展，从而延长移植物的存活时间？
目的：观察他克莫司替换环孢素A治疗慢性移植肾肾病的有效性及安全性。
设计：观察对比实验。
单位：中南大学湘雅二医院器官移植中心泌外器官移植科。
对象：选择2001–04/2005–10在中南大学湘雅二医院器官移植中心泌外器官移植科接受同种异体肾移植术后发生慢性移植肾肾病的73例患者，均经移植肾穿刺活检证实。男42例，女31例，年龄19~69岁，所有患者均对治疗项目知情同意，实验经过医院伦理委员会批准许可。环孢素A软胶囊为杭州中美华东制药有限公司或华北制药有限公司产品，吗替麦考酚酸酯胶囊购自上海罗氏制药有限公司，醋酸泼尼松片为中南大学湘雅二医院提供，他克莫司胶囊为藤泽药品中国有限公司产品。
方法：根据患者治疗意愿将患者分为环孢素A组（n =30）和他克莫司组（n =43），两组患者性别、年龄及一般情况差异无统计学意义（P > 0.05）。环孢素A组患者维持原免疫抑制方案进行治疗，即环孢素A、霉酚酸酯及泼尼松联用。他克莫司组将环孢素A转换成他克莫司， 他克莫司转换方案为停服环孢素A 24 h后开始服用他克莫司，其起始剂量为0.08~0.1 mg/（kg·d），早晚餐后2 h口服，服药3 d后测其谷值浓度，要求其血药浓度维持在5~8μg/L，并根据血药浓度、血清肌酐及其毒副作用调整剂量，其他用药同环孢素A组，两组均治疗12个月。
主要观察指标：①治疗12个月后监测两组患者血清肌酐、肾小球滤过率、24 h尿蛋白定量、血脂（包括总胆固醇、三酰甘油、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇等生化指标）变化情况；②观察药物毒副作用（震颤、高血糖及高血压等发生情况）。
结果：患者73例均进入结果分析。①生化指标检测结果：转换治疗12个月后，他克莫司组血清肌酐及24 h尿蛋白定量显著低于环孢素A组，差异有统计学意义(P < 0.01)；肾小球滤过率显著高于环孢素A组，差异有统计学意义（P < 0.01）；总胆固醇、三酰甘油、低密度脂蛋白胆固醇均显著低于环孢素A组，差异有统计学意义（P < 0.01）。②毒副作用：他克莫司组震颤发生率为，高于环孢素A组[30% (9/30)，5%（2/43），(P < 0.01)]，差异有显著性意义，高血压发生率为，显著低于环孢素A组[56%(24/43)，83%，(25/30)，(P < 0.05)]，差异有显著性意义。
结论：他克莫司转换治疗后，肾功能减退得到延缓，蛋白尿减少，血脂代谢得到改善，他克莫司可以有效延缓慢性移植肾肾病进展。
关键词：他克莫司；环孢菌素；肾移植；肾病；移植物
中图分类号: R692 文献标识码: A 文章编号: 1673-8225(2008)05-00979-04
谢续标，彭龙开，彭风华，余少杰，王彧，姜奕，蓝恭斌，方春华，聂曼华．他克莫司替换环孢素A对慢性移植肾肾病进展的影响[J].中国组织工程研究与临床康复，2008，12(5):979-982
[www.zglckf.com/zglckf/ejournal/upfiles/08-5/5k-979(ps).pdf]
（Edited by Lavjay B/Yang Y/Wang L）