Left ventricular hypertrophy (LVH) 1 year after renal transplantation is associated with reduced long-term survival and increased risk of de novo heart failure (1). Conversion from calcineurin inhibitors (CNI) to sirolimus proved to be effective in regressing LVH in both kidney and heart transplant recipients with mild renal impairment that was most likely the result of CNI nephrotoxicity (2, 3).
However, mammalian target of rapamycin (mTOR) inhibitors are associated with an increased incidence of severe side effects, and this could limit their adoption as first line immunosuppressive agents (4).
Everolimus (EVL), a proliferation-signal inhibitor (PSI) with immunosuppressive and antiproliferative effects (5), allows early CNI minimization while maintaining good immunosuppressive efficacy and preserving renal function (6).
Herein, we report the results of a randomized controlled study aimed at assessing the impact of an immunosuppressive regimen consisting of EVL plus reduced exposure cyclosporine A (CsA) on the left ventricular mass (LVM) of nondiabetic renal transplant patients who received a single kidney from a deceased donor.
The study cohort consisted of 30 consecutive nondiabetic patients who received a single kidney graft from a deceased donor at our institution between August 1, 2008, and December 31, 2009. Ten were randomized to receive EVL plus reduced exposure CsA, and 20 to standard dose CsA. Figure 1 shows the flow of the trial.
No significant differences in demographics, clinical, and echocardiographic data were found between the two groups at baseline (Table 1). There were no drop-outs in of the groups, and all patients completed the 1-year observation period.
Biopsy-proven acute rejection occurred in one patient in the EVL group and in two subjects on standard exposure CsA. Nonetheless, acute rejection did not result in graft loss in any of the three patients. Mycophenolate mofetil was discontinued due to intolerance in 1 of the 20 subjects in the control group, whereas steroid therapy was continued throughout the entire follow-up period in all patients in both groups.
A significant reduction in both systolic and diastolic blood pressure (BP) was shown after 1 year in renal transplant recipients (RTRs) who were administered standard dose CsA, whereas BP reduction did not reach statistical significance in patients receiving EVL. A comparison analysis, however, revealed no differences in BP changes between the two groups (Table 2). To achieve this BP reduction a slight, though not significant increase in the amount of antihypertensive drugs was needed in both groups during the 1 year observation period (from 1.5±0.8 to 1.7±0.9, P=0.21 in the control group; from 1.7±0.7 to 2.0±1.0, P=0.28 in the EVL group). However, no significant differences were shown between the two groups as regards both the degree of this increase (between group difference −0.1±0.27; 95% CI, −0.5 to 0.61; P=0.86) and the different classes of drugs that were administered. A significant increase in both cholesterol and triglyceride serum levels and in daily proteinuria was observed in RTRs who were randomized to EVL during the 1-year follow-up. Changes in serum lipid profile were significantly different in the EVL group as compared with controls, whereas no differences were evident between the two groups as far as the other parameters were concerned (Table 2).
The EVL group showed significant LVM index (LVMi) regression after 1 year (from 54.7±10.6 to 45.8±9.4 g/m2.7; P=0.0016; paired t test), which was mainly due to a reduction in the thickness of both the end diastolic interventricular septum thickness (IVSd) (from 11.8±1.2 to 10.7±1.7 mm; P=0.007; paired t test) and the end diastolic thickness of the left ventricular posterior wall (PWd) (from 11.3±1.6 to 10.1±1.5; P=0.037; paired t test). By contrast, LVMi remained unchanged in controls. Figure 2 shows the behavior of LVMi in the two groups during the 1-year observation period.
Changes in LVMi and both IVS and PW were significantly different in patients who were administered EVL as compared with controls (Table 2). Moreover, after 1 year, LVMi was significantly lower in the EVL group than in controls (P=0.017, unpaired t test).
Finally, the proportion of patients showing LVH regression or even renormalization was greater in the EVL group (80%) than it was in the control group (15%) (P=0.0051; chi-square analysis).
Univariate linear regression analysis showed that EVL therapy (r=0.48; P=0.0079), greater baseline LVMi (r=0.46; P=0.0098), and changes in serum cholesterol (r=0.37; P=0.047) were significantly associated with changes in LVMi after 1 year. EVL therapy and baseline LVMi remained the sole significant predictors of 1-year LVMi reduction according to a multivariate regression model adjusted for age, gender, BP changes, and baseline serum creatinine, which accounted for 49% of the total LVMi variance of our sample (P=0.0015) (Table 3).
The most important finding of this study is that an immunosuppressive regimen consisting of EVL plus reduced-exposure CsA is associated with LVH regression in RTRs. Two previous studies reported LVH regression in renal or cardiac transplant recipients after conversion from CNI to sirolimus (2, 3). However, patients enrolled in both of those studies underwent complete discontinuation of CNI and conversion to an mTOR inhibitor-based immunosuppressive protocol because of mild renal impairment related to CNI nephrotoxicity or chronic allograft dysfunction. Consequently, concerns were raised over whether mTOR per se or CNI discontinuation was the major factor inducing LVH regression. By contrast, patients randomized to EVL in this trial were de novo kidney transplant recipients who were also administered reduced exposure CsA immediately after grafting, and thus suggesting that the regression of cardiac hypertrophy could actually be the effect of the mTOR inhibitor per se, and not the result of CNI discontinuation.
Our results are fully consistent with findings from studies conducted in the animal model that showed that inhibition of the mTOR pathway by rapamycin was effective in reducing or even preventing the cardiac hypertrophy caused by pressure overload (7, 8) or by surgically induced renal injury (9). Interestingly, surgically induced renal injury mice whose BP was lowered by hydralazine showed no cardiac response compared with animals treated with the mTOR inhibitor (9). Consistent with this finding, no association was found between BP changes and reduction of LVMi in our patients, and thus ruling out that BP lowering may have played a main role in achieving LVH regression, at least in our cohort. It is noteworthy, in fact, that significant reductions in BP values which then led to adequate BP control, did not reduce the LVMi of patients randomized to standard-exposure CsA, whereas patients receiving EVL who achieved similar, adequate, BP control showed significant LVH regression. EVL proved to be the only significant predictor of LVH regression in our cohort, together with greater LVMi at baseline. Considering that no differences in baseline LVMi were observed between patients on EVL plus reduced-exposure CsA and RTRs being administered standard-dose CsA, EVL emerged as the sole factor involved in the regression of LVH in the RTRs enrolled in our cohort.
This regression was mostly the result of a reduction in left ventricle (LV) thickness of both the interventricular septum and the posterior wall, whereas no significant changes were seen in the internal size of the LV. Thus “true” hypertrophy regression was achieved, consistent with a previous report that showed that mTOR inhibition was effective in decreasing LVMi mainly by reducing the thickness of the myocardial LV wall (10). The same study also demonstrated that mTOR inhibition induced a significant reduction of myocardial interstitial fibrosis. Taking into account that myocardial interstitial fibrosis is a typical pattern associated with the LVH of renal patients (11–13), and in keeping with the observation by Gao et al., we speculate that the PSI EVL may have reduced the degree of myocardial fibrosis, thus regressing LVH, and may also have prevented its new onset or worsening because none of our patients on EVL showed an increase in LVMi during the 1-year follow-up period.
Another finding of our trial is the low incidence of acute rejection in the arm randomized to EVL. It was similar to what was observed in patients on standard CsA regimens, and consistent with the evidence emerging from previous studies conducted in renal transplant patients (14, 15). The incidence of new-onset diabetes after transplantation was the same in both groups, thus ruling out that diabetes, which can occur in RTRs on mTOR inhibitor therapy (16) may counteract the effect on patients' CV prognosis of EVL-induced cardiac hypertrophy regression.
A slight but significant increase in the urinary protein excretion rate was observed in patients randomized to EVL, and this is a relevant clinical finding in the light of the association of even low-grade proteinuria with increased cardiovascular risk profile in RTRs (17). Nonetheless, the proportion of subjects with proteinuria greater than 0.3 g/24 hr after 1 year was similar in both groups, and thus suggesting that patients administered EVL should not be exposed to additional CV risks.
Last finding is the good safety profile of EVL provided that trough levels (TL) were targeted to 3 to 8 ng/mL, which again is consistent with previous reports (18, 19). Limitations to our study are that it is a single center trial, it has a small sample size, and a short follow-up period. However, the power of this trial was calculated to predict significant LVH regression on the basis of a previous nonrandomized study carried-out by our group (2). Nonetheless, the 1-year follow-up and the small number of enrolled patients allowed us to detect significant changes in the echocardiographic parameters of our RTRs randomized to EVL. Another limitation is that the slight, although significant, reduction in LV wall thickness which translates into cardiac hypertrophy regression, could be the effect of intrapatient variability. However, echocardiographic examinations were performed by the same cardiologist, whose intraobserver variability is lower than 5%, and this should minimize the risk of incorrect measurements. A final limitation is that the use of renin-angiotensin system-blocking agents was not allowed in our study. It was an “a priori” choice aimed at ascertaining the true effect of EVL on LVMi by avoiding the use of drugs, such as angiotensin-converting enzyme inhibitors which had previously proven to regress LVH in RTRs (20, 21).
The strength of our study, by contrast, includes its nature of a randomized controlled trial, which to the best of our knowledge is the only one available to date on this topic. Another strength is that the echocardiographic studies with LVMi determination were performed by an experienced cardiologist who was blinded to the arm each patient had been assigned to.
Even though findings from our study do not definitely clarify the relative weight of PSI adoption or CNI minimization for achieving LVH regression, they raise concerns over whether immunosuppressive protocols that adopt PSI and CNI minimization could be considered a first choice option for kidney transplant candidates suffering from LVH at the time of transplantation. Recently, the effect of mTOR inhibition in achieving better LV function has been reported in the animal model (22), and this suggests the suitability of this strategy for patients such as RTRs who are at higher risk of heart failure (23).
Of course, larger randomized controlled trials are warranted to ascertain whether EVL can be considered a first choice immunosuppressive regimen not only to regress cardiac hypertrophy but also to improve the general and cardiac outcomes of kidney transplant recipients with high cardiovascular risk profiles.
MATERIALS AND METHODS
Patients aged 18 to 70 years undergoing single kidney transplantation were considered eligible for the study. Exclusion criteria were diabetes, dual kidney transplant, living-related donor transplant, kidney donated after cardiac death, and cardiac valvular abnormalities at the time of enrollment.
The primary outcome of this open-label randomized controlled trial was the 1-year change in LVMi, whereas secondary outcomes included 1- and 3-year change in renal graft function, and 1- and 3-year incidence of biopsy-proven acute rejection.
Subjects were randomly assigned to receive EVL plus reduced exposure CsA or standard exposure CsA. We planned a 1:2 randomization. This choice was made for ethical reasons, because the primary endpoint of the study, that is, LVH regression, cannot be considered a primary clinical objective of renal transplantation, and mTOR inhibitors that were administered to the active arm to achieve LVH regression are reportedly associated with an increased risk of acute rejection as compared with immunosuppressive protocols that adopt standard exposure CsA (24).
A computer-generated block randomization was adopted. Allocation was implemented using sequentially numbered, opaque sealed envelopes that were kept by an employee of the Regione Liguria Transplant Coordination Office who was not involved in the clinical trial.
Power of the Study
On the basis of a previous study by our group (2), we calculated that a sample size of 12 patients in the EVL plus reduced exposure CsA arm, and 24 in the standard CsA arm serving as controls would provide at least 90% power to detect significant LVMi reduction with a two-sided significance level of 0.05, and assuming a 20% decrease in LVMi in the EVL arm, an SD of this change of 7 g/m2.7 and a 10% drop-out rate. During the trial flow, a further amendment was made to our study. We decided that a sample size of 10 patients in the EVL plus reduced exposure CsA arm was large enough to ensure 80% power in detecting LVMi reduction with a two-sided significance level of 0.05.
All patients in both groups were also administered induction therapy, consisting of anti-IL-2 receptor monoclonal antibodies, and steroids. Patients undergoing standard CsA dose protocol were also administered mycophenolate mofetil. Titration of immunosuppressive drugs was tailored twice monthly. This was done in an attempt to keep EVL TL between 3 and 8 ng/mL and CsA TL between 75 and 125 ng/mL in the first 2 months after transplantation, and between 50 and 100 ng/mL thereafter in the group randomized to EVL plus reduced exposure CsA, whereas target CsA TL for RTRs enrolled in the control group were 150 to 300 ng/mL in the first 2 months, and 125 to 250 ng/mL thereafter.
Antihypertensive drugs that do not act on the renin-angiotensin system were administered to both groups to achieve BP values less than or equal to 130/80 mm Hg, which is the optimal BP target for RTRs according to the K-DOQI Guidelines (25). Office BP was measured quarterly and antihypertensive drugs were titrated on the basis of BP values. Echocardiography was performed according to the recommendations of the American Society of Echocardiography (26) at baseline and again after 12 months. Echotracings were analyzed by an expert cardiologist who was blinded to the immunosuppressive protocol administered to each patient.
Measurements included the end systolic and the end diastolic (EDD) diameters of the LV, the IVSd, and the PWd. All measurements were made in triplicate and then averaged. Intraobserver variability was less than 5%. The LVM was calculated from the above measurements according to the formula: LVM=0.80×1.04×[(IVSd+PWd+EDD)3)−EDD3]+0.6 g (27) and then indexed for height2.7. LVH was defined as LVMi more than 49.2 g/m2.7 and more than 46.7 g/m2.7 according to previously reported cut-off values for men and women, respectively (28). The relative wall thickness was calculated as the 2PW/EDD ratio. The percent fractional shortening (% FS) of the LV was calculated as EDD−ESD/EDD×100, where ESD is end systolic diameter.
The serum of each patient was tested for creatinine, hemoglobin, uric acid, lipids, and for CsA or EVL TL at baseline, which was considered the time of discharge from the Transplant Unit after hospitalization for grafting, and then twice monthly. Daily urinary protein excretion rate was also assessed at baseline and twice monthly. The protocol of this study conformed to the guidelines of the ethical committee of our institution. Each patient gave informed consent. The study was registered at http://www.controlled-trials.com/ISRCTN93681079.
Data were analyzed according to an “intention-to-treat” analysis, and presented as mean±SD. For continuous variables, the unpaired Student's t test was used for between group comparisons, whereas the paired t test was used for intragroup comparisons. Fisher's exact test or the chi-square test where appropriate were used to assess differences in the prevalence of noncontinuous variables in the two groups. Linear regression analysis was used to assess the significance of association between changes in LVMi and both baseline variables and their changes over the 12-month period. Variables which resulted significantly associated with outcome by univariate regression analysis were then included in a multiple regression model, which was constructed to identify the main predictors of LVM changes during the 1 year follow-up.
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