7.1 Study population
Between September 1999 and October 2011, 2837 patients with ESRD received a renal transplant at our center. As part of standard care, we perform an in-depth clinical investigation and biobanking of plasma samples of all patients who have received a renal transplant two to three months after transplantation. Patients not eligible for inclusion in the present study were either below the age of 16 years (n=78), transferred to local hospitals before ten weeks post-transplant (n=335) or suffered graft loss (n=58) or death (n=21) within the first ten weeks after transplantation (Figure 5). Of the eligible patients, 344 patients were not offered a clinical visit at ten weeks post-transplant due to understaffing at the laboratory and therefore missed blood samples and clinical measurements at this time-point. Furthermore, the amount of plasma collected was inadequate for individual fatty acid determination in another 11 patients. The remaining 1990 patients were included in the study.
7.2 Data collection and clinical endpoints
Blood was sampled in the fasting state at the clinical visit ten weeks after transplantation.
Routine blood samples were analyzed at our center, whereas some blood samples were immediately frozen and stored at -80º C. They were later sent to The Lipid Research Center, Aalborg University Hospital, Denmark for fatty acid analysis by gas chromatography (Figure 2) (163, 164). The stability of plasma phospholipid marine n-3 PUFAs and their ability to predict dietary intake of seafood is not affected by long-term freezer storage (165, 166).
Individual fatty acids were identified by their relative retention time and quantified as weight percentage (wt%) of total plasma phospholipid fatty acids. We used change in serum creatinine as a measure of decline in renal graft function over time. Fasting plasma glucose levels were assessed in whole blood samples using plasma calibrated HemoCue ABTM B-glucose Analyzer® (HemoCue, Ängelholm, Skåne, SWE) with adjustments for hematocrit levels as appropriate. Pulse wave velocity, resting heart rate, systolic blood pressure (SBP) and diastolic blood pressure (DBP) measurements have been registered since 2007 and were performed immediately after blood sampling in the morning of the clinical visit ten weeks post-transplant. The patients were instructed not to take any medication, do physical exercise or drink coffee or tea prior to the measurements, as this would influence results. Automated
oscillometric upper arm blood pressure measurements (CAS Medical Systems, Branford, CT, US) were used to obtained SBP and DBP measurements, from which the average of the two last measurements was recorded. Pulse wave velocity and resting heart rate were measured by SphygmoCor® version 8.0 (AtCor Medical, West Ryde, NSW, AUS).
Clinical and laboratory test results were entered into a database. Demographical data was retrieved from medical records and endpoint data were collected from The Norwegian Renal Registry. Death censored graft loss was defined as return to dialysis therapy or renal re-transplantation. Variables included in the multivariable Cox regression models were checked for consistency between data registered at The Norwegian Renal Registry and data retrieved from medical records. Random errors and misclassification were corrected and missing data obtained and entered into the database.
During most of the study period, the immunosuppressive regimen consisted of methylprednisolone and basiliximab induction, followed by maintenance therapy with mycophenolate, prednisolone and a calcineurin inhibitor (either cyclosporine A or tacrolimus) (167). Acute rejection episodes were treated with intravenous methylprednisolone followed by an increased dose of oral prednisolone. Statins were discontinued during the first three months after transplantation due to potential interactions with calcineurin inhibitors (81).
7.3 Statistical analysis
Patient characteristics at baseline ten weeks post-transplant were grouped according to plasma marine n-3 PUFA levels (quartiles). Differences between groups were evaluated using Mantel-Haenszel test of linear trend for categorical data, Kruskal-Wallis test for time in dialysis therapy and linear regression for other continuous variables.
Mortality rates: Patients were grouped according to recipient age (16-44 years, 45-59 years and 60-80 years) and mortality rates and mortality rate ratios between patients with high plasma marine n-3 PUFA levels (at or above the median level of 7.95 wt%) compared with low levels (< 7.95 wt%) were estimated.
Mortality and graft loss risk: We used multivariable Cox proportional hazard regression to assess associations between plasma marine n-3 PUFA, EPA and DHA levels and mortality endpoints (Paper I). A similar approach was used to assess associations with graft loss endpoints (Paper III). The observational time started at ten weeks post-transplant. Surviving
patients were censored at the 1st of February 2014. Model assumptions were checked by inspection of the log-log patient and graft survival time plots and by a formal hypothesis tests (Schoenfeld residuals). When plasma marine n-3 PUFA levels were grouped (quartiles) and death censored graft loss was the outcome, the assumption of proportional hazard was not met. Therefore, we decided that the Cox regression analysis should only be performed with plasma marine n-3 PUFA levels included as a continuous variable for graft loss endpoints.
There was complete data on all variables included in the Cox regression models in 99.3% of patients. Patients with missing data were not included in the analyses.
Linear regression: We investigated associations between plasma marine n-3 PUFA, EPA and DHA levels and cardiovascular risk markers by unadjusted, age- and gender adjusted and multivariable adjusted linear regression analysis (Paper II). In the unadjusted and age- and gender adjusted models all variables were forced into the model. In multivariate linear regression, we used p<0.10 for selection of variables in the final models, which were analyzed in a stepwise backward manner. Time in dialysis and plasma EPA levels both showed a skewed distribution. They were logarithmically transformed to obtain normal distribution.
Unstd. β-coeff. for plasma EPA levels presented in Paper II are therefore not representative of the true slope of the regression line, whereas Std. β-coeff. should give a more correct estimate of associations between plasma EPA levels and cardiovascular risk markers after the
transformation.
We used pre-defined variables in all multivariable Cox regression and linear regression models (Table 4). In addition, we found a statistical significant interaction between plasma marine n-3 PUFA levels and recipient age for mortality and functional renal grafts lost due to recipient death. We therefore included a product term (plasma marine n-3 PUFA level multiplied with recipient age) in the Cox models. This interaction had a minor impact on results regarding mortality endpoints, but was included in the final model. There was no interaction between plasma marine n-3 PUFA levels and recipient age with death censored graft loss as the outcome. This diluted the interaction’s influence on associations between plasma marine n-3 PUFA levels and overall graft loss and it became negligible.
PASW Statistics® version 17.0 (IBM, New York, NY, US) and STATA® version 13.0 (Stata Corp, College Station, TX, US) were used for the statistical analysis. A two-sided p-value of
< 0.05 was considered statistically significant.
Table 4. Pre-defined variables included in regression models
Paper I Paper II Paper III
Variables recorded at time of renal transplantation
Recipient age and gender Recipient age and gender Recipient age and gender
Donor age Donor age
Time in dialysis therapy Time in dialysis therapy Time in dialysis therapy Preemptive transplantation Preemptive transplantation Preemptive transplantation Coronary artery disease
Atherosclerotic disease
Coronary artery disease
Stroke Stroke
Peripheral vascular disease Peripheral vascular disease
Diabetes mellitus Diabetes mellitus Diabetes mellitus
Smoking status Smoking status Smoking status
Choice of calcineurin inhibitor Choice of calcineurin inhibitor Choice of calcineurin inhibitor First or previous transplant First or previous transplant Deceased or living donor Deceased or living donor
Transplant era Transplant era
Number of HLA DR mismatches Number of HLA DR mismatches Variables recorded at a clinical visit ten weeks after transplantation
Plasma marine n-3 PUFA level
Body mass index Body mass index Body mass index
Plasma albumin Plasma albumin Plasma albumin
eGFR eGFR eGFR
Plasma total cholesterol level Plasma total cholesterol level
Plasma n-6 PUFA level (model 2) Plasma n-6 PUFA level
Early rejection Early CMV infection Induction therapy Use of mTOR inhibitor
Preemptive transplantation: No previous renal transplantation or dialysis therapy. Smoking status: Current smoker, former smoker or life-long non-smoker. Choice of calcineurin inhibitor: Use of tacrolimus or cyclosporine A. Transplant era: Early era (1999-2006) or recent era (2007-2011). HLA: Human leukocyte antigen. Early rejection: Acute rejection episodes within first ten weeks after transplantation. Early CMV infection: Cytomegalovirus infection within first ten weeks after transplantation. Induction therapy: Anti-interleukin-2 receptor antibody treatment at the time of transplantation. mTOR: Mammalian target of rapamycin inhibitor.
7.4 Ethics
Informed consent was obtained from all included patients. The study was approved by the South-Eastern Regional Committee for Medical and Health Research Ethics in Norway and was performed in accordance with the Declaration of Helsinki (ClinicalTrials.gov number NCT02017990).