1 Department of Renal Medicine, St James's University Hospital, Leeds, 2 Department of Statistics, University of Leeds, Leeds and 3 Department of Transplant Surgery, Royal Liverpool and Broadgreen University Hospitals, Liverpool, UK
Correspondence and offprint requests to: Dr J. Stoves, Department of Renal Medicine, Bradford St Luke's Hospital, Little Horton Lane, Bradford BD5 0NA, West Yorkshire, UK. Email: stovesj{at}doctors.org.uk
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Abstract |
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Methods. Forty-two cyclosporin-treated renal transplant recipients were studied. Patients were included if they had a negatively sloping reciprocal of creatinine vs time (ROCT) plot for >6 months and biopsy-proven CAN. Patients were excluded if they had previously been treated with tacrolimus/mycophenolate mofetil (MMF) or their serum creatinine was >400 µmol/l. Subjects were randomly treated with either: (A) MMF/reduced dose cyclosporin [MMF for azathioprine 0.51.0 g bd; cyclosporin trough level (C0): 75100 ng/ml]; (B) tacrolimus for cyclosporin (C0: 510 ng/ml); or (C) continuation of standard therapy. Glomerular filtration rate (GFR) was measured at baseline and after 6 months.
Results. Two patients started dialysis within 6 months (one each from groups A and B). One patient in group A was intolerant of MMF, six others reported gastrointestinal symptoms and three developed anaemia. Cyclosporin dose was reduced by 24% [interquartile range (IQR): 1427%] in group A [end-of-study C0: 99 ng/ml (IQR: 90113 ng/ml)]. In group B, the end-of-study tacrolimus C0 was 7 ng/ml (59 ng/ml). The end-of-study cyclosporin C0 in group C was 163 ng/ml (145215 ng/ml). Comparison of ROCT slopes before and after intervention revealed a treatment advantage for group A (P<0.05). The GFR analysis was supportive (P = 0.05). When patients with GFR <20 ml/min/1.73 m2 at enrolment were excluded from the analysis, the treatment advantage for group A reached statistical significance (n = 27, P<0.05).
Conclusions. MMF/reduced dose cyclosporin is superior to tacrolimus-for-cyclosporin and standard dose cyclosporin in patients with CAN, at least in the short term. The cyclosporin dose reduction component is likely to be of particular importance. Other findings suggest that early intervention is beneficial.
Keywords: chronic allograft nephropathy; cyclosporin; glomerular filtration rate; mycophenolate mofetil; randomized controlled trial; tacrolimus
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Introduction |
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Mycophenolate mofetil (MMF) is a potent and specific inhibitor of purine biosynthesis and, hence, T and B lymphocyte proliferation. There is some evidence that MMF in combination with a reduced dose of cyclosporin is an effective regimen for the treatment of established CAN [24]. The reported studies did not include control patients (no change to immunosuppressive regimen) and glomerular filtration rate (GFR) was not measured by reference methods. In some studies there was no histological confirmation of CAN, patient follow-up was of variable duration and the use of concurrent therapies that may have altered the rate of progression of CAN [for example, angiotensin-converting enzyme (ACE) inhibitors and hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors] was unreported. The majority of participants in the study by Weir et al. [3] were male African-Americans, making extrapolation of the findings to European transplant populations more problematic. The addition of MMF without concomitant cyclosporin dose reduction or withdrawal has not been beneficial in all of the reported studies, but these studies are limited also by small patient numbers, variable duration of follow-up, non-reference methods of measuring transplant function and a lack of comparative control group data [58].
Pilot studies of tacrolimus in place of cyclosporin for patients with CAN have been small and uncontrolled [9,10]. In one study, 5 out of 14 cyclosporin-treated renal allograft recipients who were converted to tacrolimus showed stability or improvement of function [9].
The promising results achieved in pilot studies of immunosuppression conversion for CAN need to be confirmed by larger, more rigorous studies that are prospective, randomized and controlled. The inclusion of a control group of patients whose immunosuppressive therapy is unaltered and the use of reference isotope techniques rather than serum creatinine to estimate renal function are of particular importance. We have examined two treatment regimens that may be beneficial in cyclosporin-treated patients with CAN: MMF with reduced dose cyclosporin and tacrolimus in place of cyclosporin.
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Subjects and methods |
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Scope
The electronic records of renal transplant recipients receiving follow-up care in the participating units were interrogated to identify patients with progressive allograft dysfunction, defined by a downwards sloping reciprocal of serum creatinine vs time (ROCT) plot over a minimum period of 6 months. The suitability of these patients for study entry was then assessed in clinic.
Eligibility
The main inclusion criteria for the study were as follows: age >18 years; cyclosporin-based immunosuppression; 6 months post-renal transplant with no episodes of acute rejection during the preceding 3 months; normal renal transplant ultrasound and Doppler examination; and biopsy-proven CAN. Transplant biopsy specimens were examined by a nominated histopathologist in each unit and classified according to the Banff criteria [12]. Exclusion criteria included the following: previous treatment with tacrolimus or MMF; HIV, HBV or HCV infection; serum creatinine >400 µmol/l; pregnant or breast-feeding female patients; female patients unwilling or unable to use approved contraception; significant, uncontrolled concurrent infection; and participation in another clinical trial during the previous month.
Eligible patients were provided with a leaflet containing information regarding the background to the study, the rationale for intervention, the timetable of the study, profiles of the study medications (MMF and tacrolimus) and issues of patient consent and confidentiality of study data.
Treatment groups
The randomized treatments were as follows: (A) MMF and reduced dose cyclosporin; (B) tacrolimus in place of cyclosporin; and (C) no change (continuation of a cyclosporin-based immunosuppressive regimen). The protocol for each is summarized below.
MMF and reduced dose of cyclosporin
Tacrolimus in place of cyclosporin
Control group
No change to treatment regimen, target cyclosporin whole blood trough levels maintained as per unit protocol during the study period.
Randomization
Patients were allocated randomly to one of the three treatment groups using a computer-generated sequence. Information regarding the randomized treatment was concealed in sequentially numbered, sealed opaque envelopes. These were opened in the presence of the patient (by J.S. at SJUH/BSLH and G.O. at RLBUH) immediately after obtaining informed, written consent for participation in the study. Both patient and physician were necessarily aware of the randomized treatment in all cases, but members of staff in the various clinical laboratories (including Medical Physics) were blinded to this information.
Investigations
Baseline investigations included height, weight, blood pressure, GFR (99mTcDTPA-GFR), full blood count, renal biochemistry, glucose, HbA1c, fasting lipids, cyclosporin trough level and urine microscopy, culture and sensitivity.
99mTcDTPA-GFR measurement comprised upper limb intravenous injection of a small volume of solution containing 99m technetium of known radioactivity bound to DTPA and blood sampling from the contralateral upper limb at 2, 3 and 4 h post-injection. The degree of residual radioactive signal present in these samples was plotted on a decay curve, from which crude GFR was calculated and subsequently adjusted for body surface area using the Dubois formula [13]. All GFR measurements were performed in the mid-morning to early afternoon period.
Dosing and administration of additional medication
All treatments that were commenced during the study period were recorded, including dose and indication. ACE inhibitor and HMG-CoA reductase inhibitor (statin) medications were not added to patient treatment regimens as per study protocol.
Safety assessment
Patients were evaluated for adverse events following enrolment. An adverse event was defined as any untoward medical occurrence. A clinical examination and laboratory safety tests were performed at each clinic visit.
Study schedule
Patients were reviewed weekly for the first month, fortnightly for the second month and monthly thereafter. A second 99mTcDTPA-GFR measurement was performed at the end of the 6 month study period.
Primary study outcome measure
The primary outcome measure was change in renal function. This was assessed by comparing 99mTcDTPA-GFR values at the start of the study and at its conclusion 6 months later and the slope of ROCT plots before and after treatment intervention.
Secondary study outcome measures
Hypertension and hypercholesterolaemia, two important side effects of immunosuppressive therapy, were selected as the secondary study outcome measures. Change in urine protein creatinine index (PCI) was also assessed.
Power of study
There is a variable rate of functional decline in some patients with CAN, such that slowing of disease progression may be independent of the therapeutic intervention. Assuming a SD of 4 ml/min/1.73 m2 for the annual reduction in GFR in patients with CAN, a study population of 48 patients would be required for a 3 ml/min/1.73 m2 per year difference between treatment groups to be statistically significant (P<0.05, power 80%).
Statistical methods
The difference in slope of the pre- and post-intervention ROCT plots was calculated for each patient and an intergroup comparison of values was made using a KruskalWallis test. It was not assumed that pre- and post-study slopes would have a common intercept at time zero. Intergroup comparisons of changes in GFR, cholesterol, triglycerides and systolic and diastolic blood pressure between the start and finish of the study were also made using a KruskalWallis test. Analysis of covariance (ANCOVA) was performed to assess the relevance of the pre-study GFR to therapeutic response.
Interim analysis
An interim analysis of data was conducted after 36 patients had completed the study (75% of the required number) by a statistician who was otherwise uninvolved with the research. There were two main reasons for not performing the analysis sooner. Firstly, the differences in outcome between the three treatment groups were not expected to be large. Secondly, the study was of short duration and it was felt that the initiation of a more effective treatment (if this were to be proven) for a chronic pathology could be deferred without major consequences. The analysis was reviewed by senior clinicians who had been involved in the planning, but not the execution, of the study.
The study sponsors had no involvement in the study design, the collection, analysis and interpretation of data, the writing of the report and the decision to submit it for publication.
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Results |
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There were no differences between the three study groups at baseline that were of statistical or clinical significance. A patient flow chart for the study participants is shown in Figure 1. Two patients started dialysis treatment during the 6 month study period, one in each of the intervention groups. In both cases the initial GFR was <20 ml/min/1.73 m2 and there was evidence of a rapid pre-study decline in graft function. An end-of-study GFR of 5 ml/min/1.73 m2 was assumed for both patients in the main GFR analysis. Two patients failed to attend for an end-of-study GFR measurement (a control group patient and a patient who appeared to respond well to a MMF/reduced dose cyclosporin regimen according to serum creatinine values) and GFR was not measured in another control group patient because of her needle phobia. These patients were, therefore, not included in the GFR analysis.
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Cyclosporin dose was reduced by a median of 24% [interquartile range (IQR): 1427%] in the group receiving MMF, giving a median end-of-study cyclosporin trough blood level of 99 ng/ml (IQR: 90113 ng/ml). The median maintenance dose of MMF was 1.5 g/day (IQR: 1.52 g/day). The median end-of-study tacrolimus trough blood level in the group receiving tacrolimus was 7 ng/ml (IQR: 59 ng/ml). The median end-of-study cyclosporin trough blood level for control group patients was 163 ng/ml (IQR: 145215 ng/ml).
ROCT plots were constructed for each patient, taking creatinine data from 12 months before to 6 months after study entry. During the run-in period, serum creatinine was measured when patients attended for routine clinical review. The median number of creatinine values per patient over the run-in period was 10 (IQR: 712). A comparison of change in ROCT slope revealed a significant treatment advantage for patients in group A (KruskalWallis test, P<0.05). Dotplots for change in ROCT slope are shown in Figure 2.
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Discussion |
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Our study provides evidence of a treatment advantage for MMF/reduced dose cyclosporin as compared with substitution of tacrolimus for cyclosporin and standard dose cyclosporin in patients with established CAN, at least in the short term. A reference method of GFR measurement (99mTcDTPA-GFR) was used to monitor change in transplant function in addition to more frequent serum creatinine measurements. The 99mTcDTPA-GFR method was performed only twice (at the start of the study and 6 months later). ROCT plots gave more information about pre-study patterns of functional attrition. Analyses of GFR and creatinine data gave similar results. It would have been preferable for the number of study participants to at least equal the power calculation estimate, but this could not be achieved within the time limits of the study. It can be seen that for control patients the decline in renal function according to ROCT plots was a little slower during the study period as compared with the run-in period (Figure 2). This may have been due to a number of factors, such as improved clinical management of patients during the study period and the inherently variable rate of disease progression that has been reported in CAN [15]. These points serve to emphasize the importance of including a control group in randomized studies.
The apparent success of the above regimens could be explained simply in terms of the known effect of cyclosporin on renal haemodynamics [16] and pro-fibrotic cytokine expression [17]. MMF is effective in reversing some of the histopathological features of CAN in the rat [18] and therapeutic concentrations of the drug inhibit fibroblast growth in cell-culture studies [19]. There is also some evidence that the use of MMF in place of azathioprine produces clinical benefit in terms of a reduced incidence of chronic renal allograft failure [20]. Although the main aim of the study was to study the safety and efficacy of immunosuppression conversion for CAN rather than the mechanism by which benefit might be achieved, we did perform a post-hoc analysis comparing data from 1 year before to the start of study and data from 3 months to 1 year to determine whether the response to MMF/reduced dose cyclosporin was attributable to a step change in GFR following cyclosporin dose reduction or this and a continuing protective effect of MMF. Our basic assumption that the acute GFR response to cyclosporin dose reduction occurs within 3 months is supported by data from Dudley et al [14]. We found a non-significant improvement in ROCT slope for the 3 months to 1 year period compared with the run-in period (P = 0.08). The body of follow-up data at 12 months was less complete than at 6 months, because of graft loss in a control patient and discontinuation of tacrolimus in a patient who developed progressive hair loss.
The importance of intervening at a relatively early stage in the development of CAN is highlighted by two separate findings of this study. Firstly, baseline GFR (GFR0) was found to be a significant predictor of outcome in an ANCOVA model with GFR0 as the covariate. Secondly, an analysis of change in GFR that excluded patients with a GFR0 of <20 ml/min/1.73 m2 revealed a more definite difference in outcome between the treatment groups. These findings emphasize the need for prompt detection of CAN.
The rate of change of GFR was reduced in some patients receiving tacrolimus, but the treatment response in the tacrolimus group as a whole was not superior to controls. The characteristics of patients who appeared to respond well were not different to those of others in the group. There was a non-significant trend towards an improvement in the serum lipid profile of tacrolimus-treated patients as compared with controls.
In summary, this randomized controlled comparative study of immunosuppression regimens in patients with established chronic allograft nephropathy provides evidence to support the use of MMF and low dose cyclosporin in preference to a standard dose cyclosporin-based regimen. More substantial reductions in cyclosporin dose might produce an even better outcome, as this component of the regimen appears to have the greatest impact on graft function, at least in the short term. However, the increased risk of acute graft dysfunction associated with total cyclosporin withdrawal needs to be considered [21]. Further studies to compare MMF-based regimens with newer immunosuppressive agents, such as rapamycin, might help to determine an optimal regimen for the prevention and treatment of CAN. Timely intervention depends on early detection of CAN. This is probably best achieved by protocol transplant biopsy as there are currently no validated non-invasive tests of renal allograft injury. Control of non-immunological factors, such as blood pressure and serum lipids, remains an important treatment goal.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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