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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Background  Calcineurin inhibitor (CNI)-related nephrotoxicity significantly contributes to chronic renal failure after liver transplantation.

Methods  In this prospective study, liver transplantation patients with renal dysfunction were randomized either to receive mycophenolate mofetil (MMF) followed by stepwise reduction of CNI with defined minimal CNI-trough levels (MMF group), or to continue their maintenance CNI dose (control group). Immune monitoring was performed in a subgroup of the patients.

Results  In the MMF group (n = 50), renal function assessed by serum creatinine improved >10% in 62% of patients, was stable in 36% and deteriorated >10% in 2% after 12 months compared with baseline values. Mean serum creatinine levels (± s.d.) significantly decreased from 1.90 ± 0.44 mg/dL to 1.61 ± 0.39 mg/dL and the corresponding calculated glomerular filtration rate significantly increased from 38.8 ± 9.6 mL/min/1.73 m2 to 47.0 ± 11.8 mL/min/1.73 m2 over a 12-month follow-up period. Blood pressure and levels of liver enzymes significantly decreased. In the control group (n = 25), there were no significant changes with respect to the investigated parameters. The MMF group had significantly lower numbers of circulating cytotoxic T cells compared with the controls; whereas regulatory T cells significantly increased.

Conclusion  Combined MMF and minimal dose CNI therapy after liver transplantation is nephroprotective and may promote allograft tolerance.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

A progressively increasing life expectancy after liver transplantation (LT) leads to previously underestimated long-term metabolic complications, in particular renal insufficiency.1 Although the aetiology of kidney damage in LT patients may be multifactorial, calcineurin-inhibitor (CNI)-induced nephrotoxicity significantly contributes to the development of renal dysfunction after transplantation.2–5 Thus, the increased morbidity associated with improved long-term survival after LT is becoming a growing concern and requires improved therapeutic management to ensure good quality of life for these patients, but without increasing the risk of allograft rejection.

Mycophenolate mofetil (MMF) has been introduced in immunosuppressive protocols to increase the therapeutic efficacy as well as to reduce CNI-related nephrotoxicity.6–10 MMF is a morpholinoethyl ester prodrug of mycophenolic acid (MPA), an inhibitor of inositol-monophosphate dehydrogenase that catalyzes the rate limiting step in de novo purine biosynthesis, thereby specifically suppressing the proliferation of T- and B-lymphocytes.11 In the experimental setting, MMF has been shown to inhibit type I collagen expression transcriptionally, enhance the expression of matrix-metalloproteinase-1 and modify the migratory and functional properties of fibroblasts and thus exert direct antifibrotic activities.12 Moreover, a potential anti-atherosclerotic effect due to the immunomodulatory function of MMF has been proposed.13 Thus, MMF is not nephrotoxic and does not increase cardiovascular risk.14

Although chronic CNI-induced renal insufficiency is associated with structural changes in the kidney,15–17 an improvement in renal function can be observed in patients after lowering CNI blood levels.6–8, 18 Thus, a potentially reversible functional impairment of CNI-induced vasomotor effects on arterioles as well as direct protective effects of MMF have been discussed as relevant components.19 However, to date, it is not clear in which patients CNI-related renal dysfunction is reversible and in which morphological changes persist despite reduction or withdrawal of CNI. Studies with complete replacement of CNI with conversion to MMF have reported acute cellular rejection episodes in 0–60% of patients.20–26 Most of these studies have described an unacceptably high risk of acute rejection with MMF monotherapy. Thus, we designed a prospective, randomized, monocentric study to evaluate the effect of combined MMF and minimal dose CNI therapy in stable LT patients with renal insufficiency. In contrast to previous reports,8, 27, 28 in our study, low-dose CNI therapy was accomplished with defined minimal trough levels. In addition, immune monitoring was conducted in a subgroup of patients by flow cytometric analysis of distinct lymphocyte populations, namely cytotoxic T cells (CTL) and regulatory T cells (Treg). The latter are of particular interest because it has been shown in an animal model that MMF might promote allograft tolerance by induction of CD4+CD25+ Treg.29 To our knowledge, this is the first randomized, controlled study in LT patients that investigates the impact of MMF and minimal CNI therapy on renal function, cardiovascular risk factors and liver function with concomitant immune monitoring.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study population

Adult LT patients were eligible for the study if a) they had received a primary cadaveric or living related liver transplant at least 1 year prior to study entry; b) they had serum creatinine values >1.2 mg/dL measured on at least two successive occasions more than 1 month apart; and c) there was no evidence of cellular rejection confirmed by a recent (≤6 months) liver biopsy. Exclusion criteria included a) total white cell count ≤2 × 109/L; b) haemoglobin <9 g/dL; c) platelet count ≤70 × 109/L; d) systemic infection requiring therapy; e) pregnancy; f) decompensated liver disease; and g) a rejection episode within the last 12 months prior to randomization. Patients with native chronic renal disease [other than hepatorenal syndrome (HRS)], renal artery stenosis, or diseases of the urinary tract and/or significant proteinuria (>1 g/24 h) and/or hematuria were not included. Patients’ characteristics are shown in Table 1. HRS was diagnosed according to the International Ascites Club guidelines and after exclusion of other causes.30 At LT, mean creatinine values were 1.33 ± 0.69 mg/dL in the MMF group and 1.24 ± 0.44 mg/dL in the control group (24 of 75 patients had creatinine values >1.2 mg/dL). Thirteen patients (nine in the MMF group and four in the control group) had diagnosis of HRS; of those, all except one (who had type 1 HRS) had type 2 HRS. Resolution of HRS was defined as a decrease of serum creatinine to below 1.5 mg/dL.31 HRS resolved in seven of 13 patients within a mean time of 39.6 ± 38.3 days, with a range of 13–120 days. The remaining six patients (four in the MMF group and two in the control group) were non-resolvers. Causes for renal dysfunction at LT among non-HRS patients (n = 11) were diuretics (n = 6), volume depletion (n = 3), hypotension (n = 1) and infection (n = 1).

Table 1.   Patients’ baseline characteristics
VariableMMF group (n = 50)Control group (n = 25)
  1. Values are expressed as mean ± s.d. or percentages (unless otherwise indicated).

  2. MMF, mycophenolate mofetil; LT, liver transplantation; ALD, alcoholic liver disease; HBV, hepatitis B virus; HCV, hepatitis C virus; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; AIH, autoimmune hepatitis; LD, liver disease; FHF, fulminant hepatic failure; BMI, body mass index.

Age (years)54 ± 13.756 ± 11.2
Gender (male)35 (70%)16 (64%)
Indication for LT
 ALD14 (28%)10 (40%)
 HBV5 (10%)3 (12%)
 HCV9 (18%)5 (20%)
 HBV + HCV1 (2%)1 (4%)
 Cryptogenic4 (8%)2 (8%)
 PBC/PSC/AIH4 (8%)2 (8%)
 Drug-induced LD4 (8%)1 (4%)
 FHF2 (4%)1 (4%)
 Others7 (14%)0 (0%)
Time LT – enrollment, range (months)72.4 ± 54.9, 12–19965.0 ± 49.9, 12–169
Arterial hypertension38 (76%)21 (84%)
Diabetes mellitus18 (36%)9 (36%)
BMI26.1 ± 4.226.6 ± 5.7
Serum creatinine (mg/dL)1.90 ± 0.441.82 ± 0.62
Blood urea nitrogen (mg/dL)35.8 ± 12.331.7 ± 9.7

The study was conducted in accordance with the Declaration of Helsinki, approved by the Institutional Review Board of the University of Duisburg-Essen (IRB 03-2177), and all patients provided written informed consent before study entry. Blood pressure was recorded continuously for 24 h at baseline and at 3, 6, and 12 months after randomization. Arterial hypertension was diagnosed when a blood pressure >140/90 mmHg was recorded on two or more consecutive patients visits or when the patient was receiving antihypertensive medication. Diabetes was defined according to the American Diabetes Association diagnostic criteria.32

Therapeutic protocol

After the screening procedure, eligible consenting patients were randomized (1:2) to either continue their current CNI dose (control group) or receive combined MMF and reduced CNI therapy (MMF group). Study patients who were transplanted for cholestatic liver diseases or autoimmune-related liver disease (n = 6) received additional low-dose (2.5–5 mg/day) prednisone therapy. All other patients had no additional immunosuppressive therapy. The study design and flow chart are presented in Figure 1. At the baseline visit, MMF therapy was started at a dose of 500 mg twice a day for 2 weeks, then increased to 750 mg twice daily for 2 weeks and finally increased to 1 g twice daily. The CNI dose was then progressively tapered to achieve target trough levels of 2–4 ng/mL for tacrolimus (TAC) and 25–50 ng/mL for cyclosporine A (CSA). CSA levels were measured using monoclonal fluorescence polarization immunoassay/TDx (Abbott Laboratories, Abbott Park, IL, USA) and TAC was measured using the IMx TAC II assay (Abbott Laboratories) in our central laboratory.

image

Figure 1.  Study design and flow chart of patient disposition. Totally 91 patients were screened; 78 patients successfully completed the screening phase and 13 were withdrawn for reasons of native chronic renal disease (n = 4), thrombopenia (n = 3), systemic infection requiring therapy (n = 2), instable graft function (n = 2), anaemia (n = 1) and history of rejection episode <6 months prior to screening (n = 1). Patients were randomized (2:1) to receive mycophenolate mofetil (MMF) plus minimal calcineurin inhibitors (CNI; MMF group) or remain on monotherapeutic immunosuppression (control group) with CNI target trough levels as indicated. Three patients withdrew consent just after inclusion had no measurement of serum creatinine under study treatment and were therefore excluded from the intent-to-treat analysis. Four patients in the MMF group prematurely discontinued the study because of recurrent hepatocellular carcinoma (n = 1), recurrent infections (n = 1), severe diarrhoea (n = 1), and anaemia (n = 1). One patient in the control group had missing evaluations at months 10 and 12. CSA, cyclosporine A; TAC, tacrolimus; ITT, intention-to-treat.

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Patients had regular monitoring (normal range in parenthesis) of serum creatinine (<1.2 mg/dL), blood urea nitrogen (9.8–21 mg/dL), glucose (74–109 mg/dL), platelets (136–409 × 109/L), leucocytes (3.8–10.7 × 109/L) and red blood cell counts (4.1–5.1 Mil/μL), haemoglobin (12.3–15.3 g/dL), cholesterol (<200 mg/dL), triglycerides (<200 mg/dL), transaminases [alanine aminotransferase (ALT), <35 U/L, aspartate aminotransferase (AST), <35 U/L], bilirubin (0.3–1.2 mg/dL), γ-glutamyltranspeptidase (γ-GT, <35 U/L) and alkaline phosphatase (AP, 25–100 U/L) and clinical examinations at baseline, every 2 weeks during the first 2 months, at month 3, then every 3 months until 12 months after start of MMF therapy. Glomerular filtration rate (GFR) was calculated on the basis of abbreviated modification of diet in renal disease (MDRD) equation.33, 34 A liver biopsy was required in the 6 months prior to study enrolment.

Safety and tolerability were assessed through monitoring of adverse events (such as gastrointestinal disorders and infections) and laboratory abnormalities (such as anaemia, leucopoenia, and thrombocytopenia) at scheduled visits or when they were reported during the treatment. Patients allocated to the control group were offered combined MMF and reduced CNI therapy at study completion.

Primary and secondary endpoints

The primary composite end point was the evolution of renal function assessed by serum creatinine. Secondary end points included arterial blood pressure, calculated GFR (cGFR), levels of serum lipids and liver enzymes, haemoglobin levels, white blood cell counts, platelets, side effects, allograft and patient survival and the incidence of acute rejection at 1 year.

Immune monitoring

Peripheral blood mononuclear cells were isolated by density separation over Ficoll separating solution (Biochrom AG, Berlin, Germany) at 350 g for 30 min at 4 °C. The cells were stained for 30 min on ice with fluorescein isothiocyanate-conjugated anti-CD25 (clone M-A251), phycoerythrin (PE)-conjugated anti-CD56 (clone B159), peridinin chlorophyll protein-conjugated or allophycocyanin (APC)-conjugated anti-CD3 (clone UCHT1) and anti-CD4 (clone RPA-T4), and APC-conjugated anti-CD8 (clone RPA-T8) antibodies. Respective immunoglobulin G isotypes were used, and all antibodies were purchased from BD Biosciences (Heidelberg, Germany). Forkhead transcription factor P3 (Foxp3) intracellular staining was done with PE-conjugated anti-human FoxP3 antibody (clone PCH101) staining set (eBioscience, San Diego, CA, USA) according to the manufacturer’s protocol. Four-colour flow cytometric cell acquisition was performed using FACSCalibur cytometer (BD Biosciences, Heidelberg, Germany) and data were analysed with WinMDI 2.8 software (Joe Trotter, Scripps Research Institute, La Jolla, CA, USA).

Assessment of predictive factors

To identify factors associated with an improvement in creatinine values >10%, we included various baseline parameters [body mass index (BMI), age, gender, CSA vs. TAC, blood urea nitrogen, serum creatinine and cGFR, mean arterial blood pressure, presence of hypertension, diabetes, type of pre-existing liver disease, blood sugar levels, and cholesterol and triglyceride levels] in the univariate analysis.

Statistical analysis

The calculation of the sample size was based on expected response rates of 60% in the MMF group and 20% in the control group, with 80% power for a two-sided test using an alpha of 0.05. Additionally, the number of patients in the control group was postulated to be half as many as in the MMF group. This resulted in a calculated total sample size of 63 eligible patients, 42 patients in the MMF group and 21 in the control group.

The data analysis was conducted on an intent-to-treat basis. Continuous data were expressed as mean ± s.d. (unless otherwise indicated). Values were compared between groups using Fisher’s exact test for categorical data and the unpaired Student’s t-test or Mann–Whitney-U test for continuous variables. Friedmann test was used to compare continuous values at distinct timepoints [baseline, month 3, (month 6 only for particular values), month 12 after onset of therapy] within a patient group for the global comparison. An overall alpha = 0.05 was chosen to indicate statistical significance. If the Friedman test was significant, the Dunn test was used to compare data at months 3, (6) and 12 with the baseline data. To obtain an experiment wide significance alpha level of 0.05, alphas of 0.025 and 0.017 were chosen for two and three comparisons, respectively. Improvement in renal function was defined as reduction in creatinine value by >10% of the starting value, stabilisation (no change) as ±10% change in creatinine value, and impairment as >10% increase of creatinine value.

To identify single variables associated with response, univariate analyses were performed using logistic regression. Stepwise regression analyses with a foreword variable selection were performed to detect factor combinations for the prediction of response to CNI sparing therapy. All factors with a P value <0.05 were entered in a stepwise logistic regression model, whereas all factors with a P value >0.10 were excluded from the model. The significances of the logistic regression models were tested using likelihood ratio χ2 tests. To test the significance of each independent variable in the stepwise logistic regressions, Wald tests were conducted. A two-tailed P value <0.05 was required for measuring statistical significance. Statistics were performed using Intercooled STATA 9.1 (StataCorp LP, College Station, TX, USA). The SSPS 10.0 program was used for statistical analyses.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Baseline features

From November 2003 to November 2005, 91 liver transplant recipients with CNI-induced renal dysfunction were screened for eligibility criteria. Of these, 13 patients did not qualify for the study; four were excluded due to pretransplantation-diagnosed chronic kidney disease, three had thrombocytopenia (≤70 × 109/L), two had systemic infections necessitating drug therapy; two had unstable graft function, one patient had anaemia (<9 g/dL) and another had a rejection episode 5 months prior to screening. Totally, 78 patients were randomized. Three patients withdrew consent directly after inclusion and were not included in the intent-to-treat analysis group, defined as randomized patients with at least one assessment of serum creatinine under study treatment. The actual sample size of n = 75 (MMF group, n = 50; control group, n = 25) in the intent-to-treat study population provides a power of approximately 97%.

One patient in the control group was not present at two visits (months 10 and 12) and four patients in the MMF group were prematurely withdrawn. Comparative analysis of the patients’ baseline characteristics did not show statistically significant differences between the two groups. The highest proportion of patients was transplanted for alcoholic liver disease followed by viral infections. The MMF group and control group consisted of patients with a mean age of 54 ± 13.7 years and 56 ± 11.2 years, respectively (Table 1). Time between LT and enrollment in the overall study group ranged between 12 and 199 months.

Stages of CNI-induced kidney dysfunction were assessed according to the K/DOQI Chronic Kidney Disease (CKD) classification.35 At baseline, none of the patients had normal creatinine values or stage 1 CKD. In the MMF group, 2% (one of 50 patients) and none of the controls had mild CKD (stage 2). Most patients (n = 63) had moderate CKD (stage 3), 82% (41 of 50) in the MMF group and 88% (22 of 25) in the control group. Stage 4 CKD was evident in eight (16%) and three (12%) patients, respectively.

Immunosuppression

CNI levels and doses during the study were maintained without significant changes in the control group, whereas, as expected, there was a decrease in the MMF group with CNI reduction. CSA and TAC levels in the MMF group were 110.6 ± 24.1 ng/mL and 6.59 ± 1.59 ng/mL at baseline. When MMF was increased to 1 g twice daily, the CSA and TAC doses were progressively tapered within 3.68 ± 2.13 weeks and 3.37 ± 2.28 weeks, respectively. Consecutively, there was a reduction of 54.7% of the initial dose for CSA and 71.4% of the initial dose for TAC at month 12.

Primary end point

Changes in renal function using overall intent-to-treat analysis are shown in Figure 2a,b. The largest changes occurred at month 3. Body weight within each group and between both groups did not differ statistically significant during the whole study period. Mean creatinine values ± s.d. in the MMF group vs. controls were as follows: 1.90 ± 0.44 mg/dL vs. 1.82 ± 0.62 mg/dL at baseline; 1.68 ± 0.42 mg/dL vs. 1.73 ± 0.73 mg/dL after 3 months; 1.69 ± 0.41 mg/dL vs. 1.86 ± 1.03 mg/dL after 6 months; and 1.61 ± 0.39 mg/dL vs. 1.95 ± 0.94 mg/dL after 12 months. The changes in the MMF group, but not in the control group, were statistically significant at months 3, 6, and 12 compared with baseline.

image

Figure 2.  Evolution of serum creatinine in the (a) mycophenolate mofetil (MMF) group (n = 50) and (b) control group (n = 25) through the study period. The horizontal line in each box plot represents the median. The bottom and top edges of each box correspond to the 25th and 75th percentiles. Whiskers extend up to adjacent values represent the lowest and largest values that are not outliers. In each group, values at months 3, 6, and 12 are compared with baseline creatinine; P values are indicated in cases of statistical significance (*P < 0.001). (c) Proportions of patients in the intent-to-treat population (n = 75), for whom renal function improved (decrease of serum creatinine >10% of the baseline value), remained unchanged (±10% change of serum creatinine) or deteriorated (increase of serum creatinine >10% of the baseline value). P values are indicated in cases of statistical significance (*P < 0.001). (d) The mean percentages (±s.d.) of improvement in creatinine values measured at particular time points in both groups. In the MMF group, mean serum creatinine improved by 11% at month 3, 10% at month 6, and 14% at month 12 compared with baseline. On the contrary, in the control group, there was a slight improvement (by 3%) at first but then impaired renal function by 1% and 6% at month 6 and at the end of the study, respectively.

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In the MMF group and the control group, at month 12, serum creatinine values improved in 31 of 50 patients (62%) and in four of 25 patients (16%), respectively, remained unchanged in 18 (36%) and 13 patients (52%), respectively, and increased in one (2%) and eight patients (32%), respectively (Figure 2c). The mean percentages of renal improvement at months 3, 6, and 12 are depicted in Figure 2d.

Subgroup analyses of the MMF group and control group were performed with respect to the severity of CNI-induced renal dysfunction. In patients with CNI reduction, creatinine values at baseline compared with those after 12 months were as follows: 1.39 mg/dL vs. 1.19 mg/dL (stage 2), 1.77 ± 0.29 mg/dL vs. 1.55 ± 0.26 mg/dL (P < 0.001) (stage 3) and 2.60 ± 0.49 mg/dL vs. 1.93 ± 0.71 mg/dL (P = 0.048) (stage 4). In the control group, creatinine values at baseline compared with those after 12 months were as follows: 1.64 ± 0.18 mg/dL vs. 1.74 ± 0.49 mg/dL (P = 0.406) (stage 3) and 3.07 ± 1.23 mg/dL vs. 3.40 ± 2.06 mg/dL (P = 0.794) (stage 4). The one MMF group patient with mild kidney damage at baseline had a normal creatinine value at month 12; whereas of MMF group patients with stage 3 and stage 4 kidney damage, creatinine values normalized in six and one patient, respectively. In contrast, none of the patients in the control group had normalization of creatinine levels during the study.

Secondary endpoints

Both overall patient and graft survival rates were 100%. Regarding clinical and laboratory parameters associated with cardiovascular morbidity, there was a statistically significant decrease in systolic and diastolic blood pressure at months 3, 6 (values not shown), and 12 in the MMF group but, as expected, not in the controls (Table 2). The study had no demonstrable effect on AP, γ-GT, blood sugar values or cholesterol; triglyceride levels tended to decrease in the MMF group; however, these changes were not statistically significant (data not shown). In both study groups, the proportions of patients administered antilipid agents, antihypertensive medications, oral hypoglycemics, or insulin were similar at baseline and at month 12. Interestingly, transaminases and bilirubin levels significantly improved with MMF and minimal CNI therapy, this was apparent as early as 12 weeks after study entry. It has been demonstrated recently that hepatitis C virus (HCV) transplant recipients who were treated with MMF and CNI taper showed a significant decrease in ALT levels.36 In our study, the MMF group and the control group consisted of only nine and five HCV patients, respectively. One patient in each group was transplanted for HCV and hepatitis B virus coinfection. In the control group and in the MMF group, ALT levels at baseline were significantly higher in HCV patients compared to those with other indications. This was also seen for AST levels in the controls but not in the CNI reduction group. In the MMF group, ALT markedly decreased from 56.9 ± 36.5 U/L at baseline to 34.2 ± 18.5 U/L (P = 0.117) (HCV patients) after 12 months and from 33.5 ± 22.6 U/L to 24.6 ± 13.3 U/L (P = 0.032) (non-HCV patients). In contrast, there were no significant changes in ALT values (baseline vs. after 12 months) in the controls [60.8 ± 28.0 U/L vs. 56.7 ± 30.4 U/L (P = 0.684) (HCV patients), and 31.1 ± 25.3 U/L vs. 34.1 ± 32.8 U/L (P = 0.748) (non-HCV patients)]. Upon CNI reduction, AST decreased from 43.3 ± 24.1 U/L to 35.0 ± 27.0 U/L (P = 0.499) (HCV patients) and from 33.1 ± 20.9 U/L to 26.0 ± 22.1 U/L (P = 0.077) (non-HCV patients); whereas AST increased from 64.4 ± 39.3 U/L to 67.6 ± 35.2 U/L (P = 0.896) (HCV patients) and from 28.7 ± 18.4 U/L to 29.7 ± 22.9 U/L (P = 0.886) (non-HCV patients) in the controls. In the MMF group and the control group, there were no statistically significant differences in bilirubin values at baseline between HCV patients and those with other indications. In both MMF subgroups (HCV patients and non-HCV patients), bilirubin levels decreased from 0.94 ± 0.56 mg/dL and 0.88 ± 0.61 mg/dL at baseline to 0.78 ± 0.46 mg/dL (P = 0.501) and 0.71 ± 0.41 mg/dL (P = 0.147) after 12 months. In both corresponding subgroups of the controls, bilirubin levels were not statistically significant different at baseline compared with those at the end of the study.

Table 2.   Laboratory parameters and blood pressure values during the study (intent-to-treat population). P values from the Dunn test for comparison of data at month 3 and 12 with the baseline data within a patient group; P values between the grey marked rows as a result of the unpaired Student's t-test or Mann-Whitney-U test for comparison between both groups. *GFR was calculated using the abbreviated MDRD formula. Thumbnail image of

In low-dose CNI patients, cGFR was 38.8 ±  9.6 mL/min/1.73 m2 at baseline and significantly increased to 45.0 ± 11.4 mL/min/1.73 m2 at month 3, to 44.3 ± 10.5 mL/min/1.73 m2 at month 6, and to 47.0 ± 11.8 mL/min/1.73 m2 at month 12; whereas there were no significant changes (42.4 ± 11.4 mL/min/1.73 m2, 40.5 ± 12.6 mL/min/1.73 m2 and 39.4 ± 12.0 mL/min/1.73 m2, respectively) in the control group compared with baseline values (40.4 ± 10.9 mL/min/1.73 m2). Subgroup analyses with respect to the severity of CNI-induced renal dysfunction revealed the following results: In the only MMF patient with mild CKD, cGFR increased from 63.8 mL/min/1.73 m2 to 74.8 mL/min/1.73 m2. In the remaining MMF patients, cGFR increased in those with moderate CKD (n = 41) from 41.1 ± 6.6 mL/min/ 1.73 m2 at baseline to 48.3 ± 9.8 mL/min/1.73 m2 at month 12 (P < 0.001), and in those with severe CKD (n = 8) from 23.7 ± 4.6 mL/min/1.73 m2 to 36.7 ± 14 ml/min/1.73 m2 (P < 0.005), respectively. In control patients with stage 3 and stage 4 kidney damage, there were no significant differences between cGFR values at baseline compared with those at month 12 (42.9 ± 8.8 mL/min/1.73 m2 vs. 41.8 ± 9.2 mL/min/1.73 m2, P = 0.704, and 22.1 ± 6.7 mL/min/1.73 m2 vs. 21.8 ± 9.8 mL/min/1.73 m2, P = 0.962). In the MMF group, mean blood urea nitrogen significantly improved at week 12 (28.8 ± 11.9 mg/dL), week 24 (29.2 ± 12.1 mg/dL) and week 48 (27.3 ± 12.0 mg/dL) compared with mean baseline value (35.8 ± 12.3 mg/dL) and normalized in 18 patients (36%) vs. one patient (4%) in controls. Mean haemoglobin values decreased at month 3 with MMF treatment and recovered at month 6 (12.4 ± 1.7 g/dL); leucocyte counts in the MMF group (and the control group) did not significantly change throughout the study (data not shown). Mean platelet counts were 181.2 ± 65.4 × 109/L in the MMF group vs. 163.7 ±  70.5 × 109/L in controls at baseline; and there was no significant change during the study period (203.2 ± 66.8 × 109/L vs. 181.7 ± 65.4 × 109/L in controls at month 12).

Predictive factors for renal improvement

Univariate analysis showed a negative correlation between end of study renal function and age at study entry [P > χ2 = 0.026, odds ratio=0.947, 95% confidence interval (CI95%) = 0.899–0.999]. In the multivariate analysis, age remained a significant predictive factor. All other variables were not statistically significant in the univariate and multivariate analysis.

Adverse events

The incidence of adverse effects was not significantly different between the two groups (58% vs. 52% in controls). In the MMF group, patients were prematurely withdrawn due to recurrence of hepatocellular carcinoma (n = 1, day 103), severe diarrhoea with progressive weight loss (n = 1, day 96), recurrent infections (n = 1, day 163), anaemia (n = 1, day 102) with decrease of haemoglobin from 11.4 g/dL at baseline to 7.6 g/dL after 3 months. Creatinine levels from these four patients changed from 2.28 mg/dL, 1.39 mg/dL, 1.44 mg/dL and 1.81 mg/dL at baseline to 2.42 mg/dL, 1.55 mg/dL, 1.59 mg/dL and 1.71 mg/dL at month 12, respectively. Side effects are listed in Table 3. The incidence of opportunistic infections was not significantly different in both groups. Monitoring for asymptomatic cytomegalovirus (CMV) infection was not routinely performed in our study patients. Consequently, as laboratory diagnosis of CMV infection is not automatically associated with clinical symptoms we cannot rule out cases of asymptomatic CMV infection in our study population. However, all patients who had leucopoenia and/or non-specific symptoms (such as fever and/or malaise), respiratory or gastrointestinal complaints, or liver function abnormalities of unknown origin were tested for CMV infection using quantitative assays, such as antigenemia (CMV pp65 antigen) and quantitative CMV-PCR assays. In our study, patients who had gastrointestinal side effects gastroscopy and/or colonoscopy (depending on the symptoms) were performed including biopsies for virological and histological evaluation. However, we did not find any patient with CMV disease in our study population during the 12-month study period. Gastrointestinal disorders and infectious complications occurred more often in the MMF group. This difference, however, was not statistically significant (Table 3). Most of gastrointestinal complaints and infections were short-lived, mild and easy to treat successfully.

Table 3.   Adverse events during the study period
Adverse event; n (%)MMF group (n = 50)Control group (n = 25)P value
  1. Data presented are the number of patients who experienced a particular adverse event, expressed as a percentage of the intent-to-treat population.

  2. Values are expressed as mean ± s.d. or percentages.

  3. HCC, hepatocellular carcinoma; MMF, mycophenolate mofetil.

  4. * Cholangitis (n = 3); † Cholangitis (n = 1), herpes virus type 8 (n = 1) and vaginal fungal infection (n = 1).

Abdominal pain8 (16%)3 (12%)0.742
Vomiting and/or nausea4 (8%)1 (4%)0.659
Diarrhoea7 (14%)2 (8%)0.709
Respiratory tract infection12 (24%)3 (12%)0.359
Urinary tract infection2 (4%)2 (8%)0.600
Herpes zooster2 (4%)00.550
Herpes labialis2 (4%)00.550
Other infections3 (6%)*3 (12%)†0.394
de novo cancer00
HCC recurrence1 (2%)01.000

In all patients, full-dose MMF therapy was administered, there was no dose reduction. In four patients with gastrointestinal side effects, MMF was administered in four daily doses (4 × 500 mg/day).

Immune monitoring

Immune monitoring was performed in a subgroup of 36 patients (26 males and 10 females) at baseline, 3, 6, and 12 months after study start. Only results from baseline and month 12 are reported, because no significant differences were observed between baseline and the other time points. In the MMF group (n = 22) and the control group (n = 14), absolute leucocyte numbers were not significantly different after 12 months compared with the respective baseline values (data not shown). However, the percentages of lymphocytes dropped significantly from 27.0 ± 1.8% at baseline to 21.2 ± 1.3% (CI95% 3.469–8.159, P < 0.001) after 12 months of combined MMF and minimal dose CNI therapy; whereas no significant differences were evident in the control group (24.0 ± 3.4% and 26.4 ± 3.6%, CI95%−6.887 to 2.087, P = 0.257). The proportions of monocytes and neutrophils were similar at the particular time points in the MMF group as well as in controls. At the end of the study, the absolute numbers of CD3+CD8+ CTL, CD3+CD56+ natural killer T (NKT) cells, and CD3+CD4+ T helper cells significantly declined in the MMF group but not in the control group (Table 4). In the MMF group, the mean percentage of CD4+CD25high T lymphocytes significantly increased from 5.2 ± 0.7% at baseline to 6.8 ± 0.7% (P = 0.008) at month 12. Of note, 83.3 ± 1.2% (range: 66.4–97.3%) of these cells expressed high levels of Foxp3. Thus, despite a general decline in circulating lymphocytes in the MMF group after 12 months, the absolute numbers of CD4+CD25high Treg were preserved during the whole study period (Table 4). On the contrary, in the control group, at the end of the study, the mean percentage of CD4+CD25high Treg slightly declined from 6.4 ± 1.1% to 5.1 ± 0.6% (P = 0.179) leading to a significant decrease in absolute numbers of CD4+CD25high Treg in peripheral blood (Table 4).

Table 4.   Numbers of T cell subsets in peripheral blood in a subgroup of patients with immune monitoring
 CD3+CD8+ cells/nL (mean ± S.E.M.)CD3+CD56+ cells/nL (mean ± S.E.M.)CD3+CD4+ cells/nL (mean ± S.E.M.)CD4+CD25highFoxp3+ cells/nL (mean ± S.E.M.)
  1. CI95%, 95% confidence interval for difference of means; MMF, mycophenolate mofetil.

  2. P < 0.001 vs. baseline. † P < 0.05 vs. baseline.

MMF group (n = 22)
 Baseline  0.613 ± 0.092  0.130 ± 0.019  1.068 ± 0.099  0.062 ± 0.016
 Month 12  0.424 ± 0.055*  0.101 ± 0.017†  0.756 ± 0.085*  0.056 ± 0.010
 CI95%  0.063 – 0.315  0.006 – 0.049  0.184 – 0.441−0.010 – 0.023
Control group (n = 14)
 Baseline  0.474 ± 0.085  0.105 ± 0.019  0.645 ± 0.096  0.051 ± 0.037
 Month 12  0.530 ± 0.091  0.125 ± 0.034  0.728 ± 0.089  0.034 ± 0.008†
 CI95%−0.167 – 0.056−0.089 – 0.049−0.211 – 0.045  0.013 – 0.035

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Increasing concerns of chronic renal failure associated with long-term CNI therapy in up to 80% of LT recipients have resulted in the implementation of more or less aggressive CNI-sparing immunosuppressive regimens.3, 19, 37, 38

Various prospective and retrospective studies have shown that CNI dose reduction or discontinuation results in an improvement in renal function.8, 22, 28, 39 Results of the first prospective, randomized low-dose CNI plus MMF trial with an untreated control group have been published recently by Pageaux et al.27 In their study, CNI doses in the study group were reduced by at least 50% regardless of the baseline titre. Median CNI concentrations at baseline were 122 ng/mL in the study group but about 1.7 times higher (204 ng/mL) in the control group. No information has been given about the ranges of the CNI concentrations and whether baseline CNI concentrations were statistically significant different between the two groups. On the contrary, in our study, we defined target trough levels of 2–4 ng/mL for TAC and 25–50 ng/mL for CSA. We observed significant improvements in mean creatinine values and cGFR from month 3. At month 12, renal function improved (62%) or remained unchanged (36%) in 98% of patients in the MMF group; only one patient who had withdrawn from the study because of diarrhoea at day 96 developed an increase (12%) in serum creatinine values. Interestingly, three of four patients in the MMF group who had unresolved HRS and concomitant CNI-induced renal insufficiency post transplant showed an improvement in creatinine values upon switch to MMF and minimal dose CNI therapy.

Of all tested variables, younger age at study entry was the only predictive factor for renal improvement.

Several in vitro studies have provided evidence that inoculation of human mesangial cells with MPA, the pharmacologically active moiety of MMF, prevents mesangial cell proliferation and matrix production.40, 41 The antiproliferative properties of MPA in vitro have been confirmed in animal studies and recently in the kidney-pancreas transplant setting demonstrating that MMF significantly reduces glomerular and interstitial injury with pronounced decreases in myofibroblast infiltration and collagen III accumulation.42–46

To date, it is not clear to which extent MMF is able to reverse CNI-induced kidney damage. However, various studies comprising immune and non-immune-mediated renal disease have provided evidence that MMF is effective in reversing structural changes in the kidney.47–49

Several previous studies addressing introduction of MMF followed by CNI reduction or withdrawal did not find any change in liver enzymes during the study period.6, 27 Flares of transaminases and/or cholestasis markers have been described in various MMF mono-therapeutic regimens because of acute rejection.25, 50 Orlando et al.50 reported fluctuation of liver function tests in eight of 41 patients with presumptive episodes of acute rejection receiving 1.5 g/day of MMF and a further rejection episode in a patient while under daily dose of 2 g MMF. In our study, levels of transaminases significantly decreased throughout the study. Bahra et al.36 suggested that significantly reduced inflammation and fibrosis scores in patients receiving MMF are possibly more related to lower CNI levels than to a direct MMF effect. However, it was demonstrated previously that MMF directly exerts protective mechanisms against inflammation and fibrosis progression.51, 52 Regarding cardiovascular risk profile, data from a few prospective and retrospective studies are in agreement with our results showing that CNI reduction or withdrawal is associated with significantly decreased systolic and diastolic blood pressure values in LT patients.7, 8, 23

Upon switch to combined MMF and minimal dose CNI therapy, we did not observe any cellular rejection episodes or an increased incidence of allograft dysfunction based on standard liver chemistry tests. There are conflicting results regarding the risk of allograft rejection with withdrawal of CNI and subsequent MMF monotherapy.19, 22–24, 26 Fairbanks et al.25 reported a significant risk (19%) of severe and irreversible liver allograft rejections, which led to retransplantation in one and death in two cases. To date, individual risk estimation of acute rejection by genetic and/or immunological factors is not clinically established. Furthermore, there is no clear correlation between MPA blood concentrations and the occurrence of acute rejection episodes.50 Taken together, the present data indicate that caution should be taken when CNI therapy is replaced by MMF monotherapy, especially with respect to reported cases of severe rejections in patients that commonly reveal a stable liver graft function prior to CNI withdrawal.

Gastrointestinal disorders, which are the most important and frequent side effects associated with MMF therapy, are positively correlated to MPA levels in the blood.53 MMF-associated causes of diarrhoea may include a compromised humoral immune response under MMF therapy, a reduced proliferative activity and increased apoptosis of enterocytes, a reduced absorptive capacity caused by a higher local intestinal production of tumour growth factor-β and a local hypersensitivity reaction triggered by its acryl glucorunide metabolite.54–57 In our study, the proportion of patients experiencing gastrointestinal side effects under MMF was slightly but not significantly higher than that in the control group. Symptoms, however, were generally mild and transient, except in one case requiring withdrawal from the study. For this patient, no causative infectious agent was identified by repeated testing and the patient showed prompt resolution of symptoms after MMF was discontinued.

The underlying mechanisms of anaemia associated with the use of MMF are not fully understood. It can be speculated that bone marrow suppression may be attributed to the antiproliferative effect of MPA. However, frequently observed concomitant viral infections including Epstein-Barr and CMV may potentiate the myelosuppressive effects of MPA.58 In our study, severe anaemia occurred in one patient but resolved completely with cessation of MMF therapy.

Concomitant immune monitoring was performed in 36 of 75 patients in the intent-to-treat population. In the MMF group, at month 12, the percentage of circulating effector T cells, particularly CTL and NKT cells, was significantly lower than in the control group. These results are in accordance with previous studies showing that MPA dose-dependently inhibits the generation of CTL and prevents allograft rejection.9 As generation of antigen-specific CTL is an important immunological effector mechanism in allograft rejection, the reduction in circulating CTL might reduce the risk of allograft rejection. Human T cells expressing natural killer (NK) cell markers such as CD56 are abundant in the liver and play an important role in the host defense.59, 60 Thus, based on the tight association between cytolytic function and CD56 expression,61 the CD56 cell surface molecule is a useful marker to identify and monitor effector CD8+ T cells in the transplant setting. While the circulating effector CD8+ T cell pool diminished over time in the MMF group, there was a concomitant increase in CD4+CD25+Foxp3+ Treg, which play a key role in prevention of autoimmunity and maintenance of transplant tolerance.62, 63 Our findings are in agreement with other reports suggesting that MMF potentially may promote allograft tolerance. For instance, Gregori et al.29 indicated that MMF treatment is associated with an increased frequency of murine CD4+CD25+ Treg when combined with 1-alpha,25-dihydroxyvitamin D3. Moreover, recent evidence in human transplant recipients indicates that CNI treatment reduces the percentages of circulating CD4+CD25+ Treg.64 Therefore, it is interesting to note that Treg function seems to depend critically on calcineurin-dependent interleukin-2 production65 and that CNI interferes with Treg induction in a dose-dependent manner.66

In conclusion, our study has established the clinical safety and efficacy of combined MMF and minimal CNI therapy, as documented by progressive improvement in renal function, blood pressure profile and liver enzymes. Our findings also indicate that this combined immunosuppressive regimen may promote allograft tolerance.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Declaration of personal interests: None. Declaration of funding interests: This work was supported by an unrestricted grant from Hoffmann-La Roche, Grenzach-Wyhlen, Germany.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Sethi A, Stravitz RT. Review article: medical management of the liver transplant recipient - a primer for non-transplant doctors. Aliment Pharmacol Ther 2007; 25: 22945.
  • 2
    Liptak P, Ivanyi B. Primer: histopathology of calcineurin-inhibitor toxicity in renal allografts. Nat Clin Pract Nephrol 2006; 2: 398404.
  • 3
    Fisher NC, Nightingale PG, Gunson BK, Lipkin GW, Neuberger JM. Chronic renal failure following liver transplantation: a retrospective analysis. Transplantation 1998; 66: 5966.
  • 4
    Fung J, Kelly D, Kadry Z, Patel-Tom K, Eghtesad B. Immunosuppression in liver transplantation: beyond calcineurin inhibitors. Liver Transpl 2005; 11: 26780.
  • 5
    Monsour HP Jr, Wood RP, Dyer CH, Galati JS, Ozaki CF, Clark JH. Renal insufficiency and hypertension as long-term complications in liver transplantation. Semin Liver Dis 1995; 15: 12332.
  • 6
    Jain A, Vekatramanan R, Eghtesad B, et al. Long-term outcome of adding mycophenolate mofetil to tacrolimus for nephrotoxicity following liver transplantation. Transplantation 2005; 80: 85964.
  • 7
    Kornberg A, Kupper B, Hommann M, Scheele J. Introduction of MMF in conjunction with stepwise reduction of calcineurin inhibitor in stable liver transplant patients with renal dysfunction. Int Immunopharmacol 2005; 5: 1416.
  • 8
    Cantarovich M, Tzimas GN, Barkun J, Deschenes M, Alpert E, Tchervenkov J. Efficacy of mycophenolate mofetil combined with very low-dose cyclosporine microemulsion in long-term liver-transplant patients with renal dysfunction. Transplantation 2003; 76: 98102.
  • 9
    Allison AC, Eugui EM. Mechanisms of action of mycophenolate mofetil in preventing acute and chronic allograft rejection. Transplantation 2005; 80: S18190.
  • 10
    Klupp J, Pfitzmann R, Langrehr JM, Neuhaus P. Indications of mycophenolate mofetil in liver transplantation. Transplantation 2005; 80: S1426.
  • 11
    Allison AC, Eugui EM. Mycophenolate mofetil and its mechanisms of action. Immunopharmacology 2000; 47: 85118.
  • 12
    Roos N, Poulalhon N, Farge D, Madelaine I, Mauviel A, Verrecchia F. In vitro evidence for a direct anti-fibrotic role of the immunosupressive drug mycophenolate mofetil. J Pharmacol Exp Ther 2007; 321: 5839.
  • 13
    Van Leuven SI, Kastelein JJ, Allison AC, Hayden MR, Stroes ES. Mycophenolate mofetil (MMF): firing at the atherosclerotic plaque from different angles? Cardiovasc Res 2006; 69: 3417.
  • 14
    Boots JM, Christiaans MH, Van Hooff JP. Effect of immunosuppressive agents on long-term survival of renal transplant recipients: focus on the cardiovascular risk. Drugs 2004; 64: 204773.
  • 15
    Johnson DW, Saunders HJ, Johnson FJ, Huq SO, Field MJ, Pollock CA. Fibrogenic effects of cyclosporin A on the tubulointerstitium: role of cytokines and growth factors. Exp Nephrol 1999; 7: 4708.
  • 16
    Johnson DW, Saunders HJ, Johnson FJ, Huq SO, Field MJ, Pollock CA. Cyclosporin exerts a direct fibrogenic effect on human tubulointerstitial cells: roles of insulin-like growth factor I, transforming growth factor beta1, and platelet-derived growth factor. J Pharmacol Exp Ther 1999; 289: 53542.
  • 17
    Myers BD, Ross J, Newton L, Luetscher J, Perlroth M. Cyclosporine-associated chronic nephropathy. N Engl J Med 1984; 311: 699705.
  • 18
    Beckebaum S, Cicinnati VR, Klein CG, et al. Impact of combined mycophenolate mofetil and low-dose calcineurin inhibitor therapy on renal function, cardiovascular risk factors, and graft function in liver transplant patients: preliminary results of an open prospective study. Transplant Proc 2004; 36: 26714.
  • 19
    Barkmann A, Nashan B, Schmidt HH, et al. Improvement of acute and chronic renal dysfunction in liver transplant patients after substitution of calcineurin inhibitors by mycophenolate mofetil. Transplantation 2000; 69: 188690.
  • 20
    Schlitt HJ, Barkmann A, Boker KH, et al. Replacement of calcineurin inhibitors with mycophenolate mofetil in liver-transplant patients with renal dysfunction: a randomised controlled study. Lancet 2001; 357: 58791.
  • 21
    Stewart SF, Hudson M, Talbot D, Manas D, Day CP. Mycophenolate mofetil monotherapy in liver transplantation. Lancet 2001; 357: 60910.
  • 22
    Herrero JI, Quiroga J, Sangro B, et al. Conversion of liver transplant recipients on cyclosporine with renal impairment to mycophenolate mofetil. Liver Transpl Surg 1999; 5: 41420.
  • 23
    Moreno Planas JM, Cuervas-Mons MV, Rubio GE, et al. Mycophenolate mofetil can be used as monotherapy late after liver transplantation. Am J Transplant 2004; 4: 16505.
  • 24
    Raimondo ML, Dagher L, Papatheodoridis GV, et al. Long-term mycophenolate mofetil monotherapy in combination with calcineurin inhibitors for chronic renal dysfunction after liver transplantation. Transplantation 2003; 75: 18690.
  • 25
    Fairbanks KD, Thuluvath PJ. Mycophenolate mofetil monotherapy in liver transplant recipients: a single center experience. Liver Transpl 2004; 10: 118994.
  • 26
    Pierini A, Mirabella S, Brunati A, Ricchiuti A, Franchello A, Salizzoni M. Mycophenolate mofetil monotherapy in liver transplantation. Transplant Proc 2005; 37: 26145.
  • 27
    Pageaux GP, Rostaing L, Calmus Y, et al. Mycophenolate mofetil in combination with reduction of calcineurin inhibitors for chronic renal dysfunction after liver transplantation. Liver Transpl 2006; 12: 175560.
  • 28
    Reich DJ, Clavien PA, Hodge EE. Mycophenolate mofetil for renal dysfunction in liver transplant recipients on cyclosporine or tacrolimus: randomized, prospective, multicenter pilot study results. Transplantation 2005; 80: 1825.
  • 29
    Gregori S, Casorati M, Amuchastegui S, Smiroldo S, Davalli AM, Adorini L. Regulatory T cells induced by 1 alpha,25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance. J Immunol 2001; 167: 194553.
  • 30
    Arroyo V, Gines P, Gerbes AL, et al. Definition and diagnostic criteria of refractory ascites and hepatorenal syndrome in cirrhosis. International Ascites Club. Hepatology 1996; 23: 16476.
  • 31
    Salerno F, Gerbes A, Gines P, Wong F, Arroyo V. Diagnosis, prevention and treatment of the hepatorenal syndrome in cirrhosis. A consensus workshop of the international ascites club. Gut 2007; in press.
  • 32
    Gabir MM, Hanson RL, Dabelea D, et al. The 1997 American Diabetes Association and 1999 World Health Organization criteria for hyperglycemia in the diagnosis and prediction of diabetes. Diabetes Care 2000; 23: 110812.
  • 33
    Levey AS, Greene T, Kusek JW, Beck GJ, MDRD Study Group. A simplified equation to predict glomerular filtration rate from serum creatinine. Am Soc Nephrol 2000; 11: A0828.
  • 34
    Levey AS, Coresh J, Greene T, et al. Expressing the Modification of Diet in Renal Disease Study equation for estimating glomerular filtration rate with standardized serum creatinine values. Clin Chem 2007; 53: 76672.
  • 35
    National Kidney Foundation. K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis 2002; 39: S1266.
  • 36
    Bahra M, Neumann UI, Jacob D, et al. MMF and calcineurin taper in recurrent hepatitis C after liver transplantation: impact on histological course. Am J Transplant 2005; 5: 40611.
  • 37
    Pawarode A, Fine DM, Thuluvath PJ. Independent risk factors and natural history of renal dysfunction in liver transplant recipients. Liver Transpl 2003; 9: 7417.
  • 38
    Backman L, Reisaeter AV, Wramner L, Ericzon BG, Salmela K, Brattstrom C. Renal function in renal or liver transplant recipients after conversion from a calcineurin inhibitor to sirolimus. Clin Transplant 2006; 20: 3369.
  • 39
    Pfitzmann R, Klupp J, Langrehr JM, et al. Mycophenolatemofetil for immunosuppression after liver transplantation: a follow-up study of 191 patients. Transplantation 2003; 76: 1306.
  • 40
    Baer PC, Gauer S, Hauser IA, Scherberich JE, Geiger H. Effects of mycophenolic acid on human renal proximal and distal tubular cells in vitro. Nephrol Dial Transplant 2000; 15: 18490.
  • 41
    Dubus I, Vendrely B, Christophe I, et al. Mycophenolic acid antagonizes the activation of cultured human mesangial cells. Kidney Int 2002; 62: 85767.
  • 42
    Ziswiler R, Steinmann-Niggli K, Kappeler A, Daniel C, Marti HP. Mycophenolic acid: a new approach to the therapy of experimental mesangial proliferative glomerulonephritis. J Am Soc Nephrol 1998; 9: 205566.
  • 43
    Penny MJ, Boyd RA, Hall BM. Mycophenolate mofetil prevents the induction of active Heymann nephritis: association with Th2 cytokine inhibition. J Am Soc Nephrol 1998; 9: 227282.
  • 44
    Van Bruggen MC, Walgreen B, Rijke TP, Berden JH. Attenuation of murine lupus nephritis by mycophenolate mofetil. J Am Soc Nephrol 1998; 9: 140715.
  • 45
    Badid C, Vincent M, McGregor B, et al. Mycophenolate mofetil reduces myofibroblast infiltration and collagen III deposition in rat remnant kidney. Kidney Int 2000; 58: 5161.
  • 46
    Nankivell BJ, Wavamunno MD, Borrows RJ, et al. Mycophenolate mofetil is associated with altered expression of chronic renal transplant histology. Am J Transplant 2007; 7: 36676.
  • 47
    Badid C, Desmouliere A, Laville M. Mycophenolate mofetil: implications for the treatment of glomerular disease. Nephrol Dial Transplant 2001; 16: 17526.
  • 48
    Choi MJ, Eustace JA, Gimenez LF, et al. Mycophenolate mofetil treatment for primary glomerular diseases. Kidney Int 2002; 61: 1098114.
  • 49
    Sommerer C, Hergesell O, Nahm AM, et al. Cyclosporin A toxicity of the renal allograft--a late complication and potentially reversible. Nephron 2002; 92: 33945.
  • 50
    Orlando G, Baiocchi L, Cardillo A, et al. Switch to 1.5 grams MMF monotherapy for CNI-related toxicity in liver transplantation is safe and improves renal function, dyslipidemia, and hypertension. Liver Transpl 2007; 13: 4654.
  • 51
    Raisanen-Sokolowski A, Vuoristo P, Myllarniemi M, Yilmaz S, Kallio E, Hayry P. Mycophenolate mofetil (MMF, RS-61443) inhibits inflammation and smooth muscle cell proliferation in rat aortic allografts. Transpl Immunol 1995; 3: 34251.
  • 52
    Blaheta RA, Leckel K, Wittig B, et al. Mycophenolate mofetil impairs transendothelial migration of allogeneic CD4 and CD8 T-cells. Transplant Proc 1999; 31: 12502.
  • 53
    Brunet M, Cirera I, Martorell J, et al. Sequential determination of pharmacokinetics and pharmacodynamics of mycophenolic acid in liver transplant patients treated with mycophenolate mofetil. Transplantation 2006; 81: 5416.
  • 54
    Zmonarski SC, Boratynska M, Madziarska K, et al. Mycophenolate mofetil severely depresses antibody response to CMV infection in early posttransplant period. Transplant Proc 2003; 35: 22056.
  • 55
    Papadimitriou JC, Cangro CB, Lustberg A, et al. Histologic features of mycophenolate mofetil-related colitis: a graft-versus-host disease-like pattern. Int J Surg Pathol 2003; 11: 295302.
  • 56
    Neerman MF, Boothe DM. A possible mechanism of gastrointestinal toxicity posed by mycophenolic acid. Pharmacol Res 2003; 47: 5236.
  • 57
    Shipkova M, Voland A, Grone HJ, Armstrong VW, Oellerich M, Wieland E. Relationship between plasma concentrations of mycophenolic acid (MPA) and its acyl glucuronide (AcMPAG) and intestinal damage in wistar rats treated with mycophenolate mofetil (MMF). Ther Drug Monit 2002; 25: 537.
  • 58
    Al-Uzri A, Yorgin PD, Kling PJ. Anemia in children after transplantation: etiology and the effect of immunosuppressive therapy on erythropoiesis. Pediatr Transplant 2003; 7: 25364.
  • 59
    Satoh M, Seki S, Hashimoto W, et al. Cytotoxic gammadelta or alphabeta T cells with a natural killer cell marker, CD56, induced from human peripheral blood lymphocytes by a combination of IL-12 and IL-2. J Immunol 1996; 157: 388692.
  • 60
    Ohkawa T, Seki S, Dobashi H, et al. Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells. Immunology 2001; 103: 28190.
  • 61
    Pittet MJ, Speiser DE, Valmori D, Cerottini JC, Romero P. Cutting edge: cytolytic effector function in human circulating CD8+ T cells closely correlates with CD56 surface expression. J Immunol 2000; 164: 114852.
  • 62
    Sakaguchi S. Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004; 22: 53162.
  • 63
    Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance. Nat Rev Immunol 2003; 3: 199210.
  • 64
    Segundo DS, Ruiz JC, Izquierdo M, et al. Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+FOXP3+ regulatory T cells in renal transplant recipients. Transplantation 2006; 82: 5507.
  • 65
    Zeiser R, Nguyen VH, Beilhack A, et al. Inhibition of CD4+CD25+ T-cell function by calcineurin-dependent interleukin-2 production. Blood 2006; 108: 3909.
  • 66
    Shibutani S, Inoue F, Aramaki O, et al. Effects of immunosuppressants on induction of regulatory cells after intratracheal delivery of alloantigen. Transplantation 2005; 79: 90413.