1 Nephrology Section, 2 Research Unit, 3 Biochemical Section and 4 Service of Nuclear Medicine, University Hospital of the Canary Islands, La Laguna, Tenerife, Spain
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Abstract |
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Methods. In a double-blind clinical trial, 86 subjects with no immunological risk randomly received either 6 g/day of fish oil (fish oil group; n=46) or soy oil (control group; n=40) during the first 3 months after transplantation. The mRNA expression of interleukins (TNF-, IL-1ß and IL-2) was determined by RT-PCR using fine-needle aspiration during follow-up (at baseline and the 1st, 2nd and 3rd month after renal transplantation), as well as during acute rejection episodes and after anti-rejection therapy. The glomerular filtration rate was determined at baseline, and at 1 and 3 months post-graft by [51Cr]EDTA clearances.
Results. The incidence of acute rejection during the first post-transplant year was similar in both groups (44 vs 47%), as was 1-year graft survival (86 vs 89%). There were no differences between groups in overall renal expression of interleukins in patients who did not suffer rejections during the study. At rejection episodes, the fish oil group showed a trend toward a lower renal expression of TNF- (3.7±6.8 vs 15±18.6 TNF-
/actin, ratio of arbitrary optical units; P=0.05). In addition, a trend toward a lower IL-1ß expression after therapy was observed in the fish oil group (49.3±54 vs 84.4±59 IL-1ß/actin, ratio of arbitrary optical units; P=0.05). However, the severity of acute rejections (Banff criteria) as well as renal function after anti-rejection treatment were similar in both groups. Finally, a greater reduction in triglyceride levels was observed in the fish oil group compared with the control group (-6.6±52.7 vs 12.7±40.2%; P<0.05).
Conclusions. Treatment with fish oil during the first 3 months post-transplantation does not influence acute rejection rate and has no beneficial effect on renal function or graft survival.
Keywords: cytokines; fine needle aspiration; kidney transplantation; omega-3 polyunsaturated fatty acids
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Introduction |
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Dietary fish oil, rich in omega-3 polyunsaturated fatty acids (-3 PUFA), may have immunomodulatory effects. Indeed, a fish-oil-induced change in the cyclooxygenase and lipoxygenase pathways has been associated with alterations in leukocyte function, as well as with reduced TNF and IL-1 synthesis [1012]. In addition,
-3 PUFA suppresses IL-2 ex vivo production by mononuclear cells in healthy individuals [13]. These effects could well explain the lower incidence of previously reported acute rejections and better renal function in renal transplant patients receiving this compound [14,15]. However, little is known about the effect of fish oil on the in situ expression of interleukines after renal transplantation.
This study was undertaken, therefore, to investigate the effect of fish oil supplementation on the incidence of acute rejection during the first 3 months after renal transplantation. In addition, its effects on the in situ expression of interleukines (TNF-, IL-1ß and IL-2) as well as on renal function and other clinical variables were analysed.
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Subjects and methods |
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Patients
Study participants were consecutive first recipients of a cadaveric renal graft. Patients were eligible for inclusion in this study if they met the following criteria: (i) they were between 18 and 70 years of age; (ii) they had no treatment with fish oil or immunosuppressive therapy in the 6 months prior to the study; and (iii) they had no haemorrhagic disorders. Exclusion criteria were: (i) any investigational drug used within the past 3 months; (ii) acute liver disease; (iii) a history of malignancy within the past 2 years; (iv) fish or iodine allergy; and (v) pregnancy or lactation. Patients were also excluded if they experienced severe side effects during the follow-up.
This study was approved by the Ethics Committee of the Hospital Universitario, and by the Dirección General de Farmacia (Spanish Ministry of Health, Madrid). The study was conducted according to the Declaration of Helsinki, and each patient gave written informed consent.
Study objectives
The primary end-point of the study was to compare the effect on the incidence of acute rejection of dietary supplementation with fish oil or with soy oil during the first 3 months after renal transplantation.
Secondary objectives were to study the effect of fish oil on the in situ expression of interleukines (TNF-, IL-1ß and IL-2) as well as on renal function and other clinical and biochemical parameters.
Study design
After screening, all eligible patients were randomly assigned to receive either 6 g of fish oil (21% eicosopentanoic acid (C20:5-3) and 11% docosahexanoic acid (C22:6
-3) as their ethyl esters, with 1 IU vitamin E/g of fish oil as antioxidant; Epaleo®) or 6 g of soy oil (48% C18:2
-6, 6.5% C22:6
-3, and 1 IU vitamin E/gram of soy oil) daily for 3 months. The oils were administered in capsules, each of which contained 0.5 g of oil (12 capsules per day per patient in either group) as previously reported [14]. The administration of oil began 2 days post-operation, and after 3 months was stopped. All patients were instructed to take the medication at the same time of day to facilitate compliance. In addition, compliance was verified by pill counting at each clinic visit. The patients were also instructed to make no change to their diets during the study period. The capsules of fish oil and soy oil were identical in appearance and were provided by Byk Leo S.L. (Madrid, Spain).
Patients were questioned about adverse events at each visit. Serious adverse events were defined as any major morbid event or hospital admission for any reason, or any abnormal laboratory result associated with signs or symptoms or requiring treatment. Withdrawal from the study could result from a serious event, or if the patient wished to.
Immunosuppression, and diagnosis and treatment of rejection
Both groups received quadruple sequential immunosuppressive therapy as reported previously [16]: briefly, a 7-day course of anti-thymocyte globuline (ATGAM; Upjohn, Kalamazoo, MI, USA) for induction, and prednisone, cyclosporine and azathioprine for maintenance. The dose of prednisone was 0.3 mg/kg of body weight (bw) per day during the first 3 months, after which it was gradually reduced to 10 mg/day for 1 year. Cyclosporine (Sandimmun®; Sandoz Pharma, Basel, Switzerland) was started when plasma creatinine was <3 mg/dl, at a dose of 8 mg/kg bw/day to achieve initial trough concentrations of 250350 ng/ml. This dose was subsequently tapered and adjusted according to total blood levels. The dose of azathioprine was 1.251.50 mg/kg/day. Delayed renal graft function was defined as a urine volume <1000 cc in the absence of a fall in serum creatinine concentration and with optimal hydration. Rejection episodes were defined by clinical diagnosis and were confirmed in all patients by a percutaneous renal biopsy (n=31) or by fine-needle aspiration (FNAP) (in four patients receiving fish oil and four on soy oil) before the start of anti-rejection treatment. All biopsies and aspiration cytologies were reviewed by a single renal pathologist (C.G.), who had no knowledge of the clinical data, and who followed the criteria proposed in the Banff 93 working classification [17]. Initially, acute rejection was treated with methylprednisolone 500 mg i.v. for 3 consecutive days. Steroid-resistant rejections were treated with a 10-day course of OKT3 (5 mg/day; Ortho Pharmaceutical, NJ, USA).
Concomitant medication and treatment of hypertension
The patients were asked to avoid aspirin and other anti-inflammatory drugs during follow-up. Patients with hypertension received antihypertensive agents (beta-blockers, calcium channel blockers or alpha-adrenergic antagonists), in combination with a diuretic, if necessary, for blood pressure control, as in standard clinical practice.
Clinical and biochemical data
Clinical and biochemical data were obtained at the randomization visit, and every 15 days thereafter. Initially, evaluation of renal function was performed every day, and 24-h creatinine clearance was calculated.
Laboratory measurements
Serum creatinine and other biochemical parameters (uric acid, glucose and complete blood count) were measured by means of a computerized autoanalyser (Hitachi 717; Boehringer Manheim, Germany). Total blood cyclosporine levels were quantified by Fluorescence Polaryzed Immunoassay (FPIA) using a monoclonal antibody (Abbot, IL, USA).
Measurement of renal function with [51Cr] EDTA clearance
The glomerular filtration rate (GFR) was measured with [51Cr]EDTA clearances at baseline (once serum creatinine was <3 mg/dl) and at months 1 and 3 post-graft. Briefly, after an intravenous bolus injection of 100 µCi [51Cr]EDTA, venous blood samples were drawn between 180 and 300 min at time intervals of 30 min [18]. The clearance value (E1) was calculated as the ratio between the injected dose and the total area under the monoexponential curve determined from the radioactivity of the plasma samples. Since this calculation can overestimate the clearance (E1), we used the correction proposed by Bröchner-Mortensen et al. [19]. The day-to-day coefficient of variation was <5%.
Fine-needle aspiration
FNAP was performed 2448 h after grafting (baseline) and at months 1, 2 and 3 thereafter. In addition, FNAP was obtained as soon as graft dysfunction was detected, and before anti-rejection therapy was instituted. Further FNAP was performed 2 weeks after anti-rejection therapy.
FNAP was performed according to previously published protocols [20,21]. Briefly, a 25G needle was used to aspirate samples of 50 µl from the allograft and introduce them into 3 ml of cell culture media. A similar amount of blood (
50 µl) was obtained from the fingertip. In order to assure that the sample was representative of the allograft infiltrate, only those FNAPs that showed seven or more tubular cells per 100 inflammatory cells were included in this study. Cytospins were obtained from FNAPs and blood samples, and processed for May-GrunwaldGiemsa staining. The corrected increment was calculated as reported previously [22]. A corrected increment of more than two was considered suggestive of acute rejection.
Reverse transcriptionpolymerase chain reaction (RT-PCR) and quantification of mRNA
FNAPs with seven or more tubular cells per 100 inflammatory cells were processed for RNA extraction. RT-PCR assays were performed essentially as described [6], with minor modifications. The primers used spanned one or various introns in the gene, and no amplification of genomic DNA was detected under the conditions used. The total RNA purified from the FNAP material (1 µg) was reverse transcribed with AMV-RTase (Promega, Madison, WI, USA), and one tenth of the resulting cDNA was used as a template for ß-actin. The protocols were optimized using tonsil total RNA to assure a linear range of amplification intensity depending on the amount of cDNA used. Thus, the number of PCR cycles was kept to 25 for ß-actin and 28 for the various cytokines. PCR products were analysed by electrophoresis in 5% acrylamide, stained with ethidium bromide and observed under UV light. A digital image analysis system (Phoenix Technologies, Seattle, WA, USA) was used to assign numerical values of optical density to the amplified bands. The amplification of ß-actin mRNA, which was performed in parallel, was used as the control to which the optical density values of the various cytokine bands were referred as percentages. The use of RT-PCR to estimate relative changes in gene expression has been widely accepted since it was first proposed by Rappolee et al. [23]. Previously, we have performed control experiments that show reproducible changes in gene expression following in vitro stimulation of lymphocyte cultures.
Statistical analysis
In accordance with our experience, the incidence of acute rejection during the period studied was 40%. In a previous report, on the other hand, fish-oil-treated patients had an incidence of acute rejection around 20% [14]. Thus, a sample size of 90 patients was calculated to be the minimum to detect a difference of at least 20% in acute rejection rate by fish oil supplementation with a two-tailed
=0.05 and a power of 0.80 plus an estimated drop-out rate of 10%. All categorical variables were analysed using the chi-square test or Fisher's exact probability test (two-tailed) as appropriate. The Friedman two-way analysis of variation (ANOVA) by rank test was used to compare repeated measurements during the study. Intra-group pairwise comparisons were performed using the Wilcoxon signed-rank test with adjusted degrees of freedom. We used t-test or univariate comparisons of numerical variables. Graft and patient survival curves were calculated using the KaplanMeier method, and survival rates of the different groups were compared using the log-rank test.
All data are expressed as mean±SD and a P value <0.05 was considered significant. Computations were made using the SPSS 9.0 for Windows® statistical package (SPSS Inc., Chicago, IL).
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Results |
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All patients who had surviving allografts at any time during the study (n=85) were included in the final analysis of interleukin expression and renal function.
No major adverse events were observed, and the only complaint from patients was a fishy aftertaste. In addition, there were no clinically observed bleeding episodes nor any prolonged episode of haematuria following graft biopsies or FNAPs.
The characteristics of the recipients and donors are shown in Table 1. The two groups were comparable, except that patients on fish oil had higher maximum panel-reactive titres and pre-transplant blood transfusions, as well as a longer time on haemodialysis. Furthermore, a larger number of diabetics was observed in the control group. Table 2
summarizes clinical and biochemical data during follow-up. As expected, a significant decrease in triglyceride levels was observed in the fish oil group. The mean 24-h urinary protein excretion levels at month 3 were similar in both groups, as were mean blood pressure and renal function. The overall incidence of acute rejection during the first post-transplant year was similar in both groups (44 vs 47.5%). Most of these acute rejections occurred during the first month after grafting (Table 2
). Episodes of acute rejection per patient were similar in both groups, and rejections were also histologically similar according to Banff criteria (fish oil group: 3 I, 8 IIA, 4 IIB and 1 III; control group: 4 I, 7 IIA, 2 IIB and 2 III). In addition, the incidence of steroid-resistant rejection was similar in both groups (13 vs 10%). Finally, a similar incidence of delayed renal function and acute cyclosporine toxicity episodes were observed in both groups (Table 2
). The dose and whole-blood cyclosporine trough concentrations of the two groups did not differ throughout the follow-up period (Table 2
).
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Renal function data
Overall, renal function data did not differ between the two groups during follow-up (Table 5). In addition, the proteinuria and renal function of patients who suffered acute rejections were similar during the study, both at rejection diagnosis and after treatment (2 weeks later) (Table 6
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Outcome
Seventy-three patients completed the first year after randomization. One year graft survival was 86 and 89% in fish oil group and control group, respectively (P=not significant (NS)).
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Discussion |
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Since the highest risk of suffering acute rejection is during the first 3 months after transplantation, these substances were administered during this period. No drug-related side effects were observed in the cohort completing the study. Additionally, there was no clinical bleeding following FNAPs.
To our knowledge this is the first randomized, placebo-controlled study to use FNAP to investigate the effect of -3 PUFA on in situ gene expression of interleukins in renal transplant patients. FNAP may be helpful to detect the production of IL-1ß, TNF-
and IL-2 mRNA, providing evidence that these factors are produced in situ in allograft transplantation, as has been demonstrated in previous reports [6,24]. Moreover, the standard FNAP technique has proven useful in differentiating acute cellular rejection from other causes of graft dysfunction [21,22].
Dietary fish oil seems to have immune-modulating effects [25,26]. This notion is concordant with the effects of fish oil on the cyclooxygenase and lipoxygenase pathways, and the resulting changes in the generation of eicosanoids; all important compounds in the sequence of renal allograft rejection. A fish-oil-induced change in the leukotriene profile has been associated with a marked reduction in the chemotactic properties of leukocytes and with a reduced production of TNF- and IL-1ß [1012]. In addition,
-3 PUFA n-fatty acids suppress interleukine-2 production and mononuclear cell proliferation in healthy volunteers [13]. These effects have been also attributed to a decreased formation of PGE2 and LTB4, although an increase of trienoid prostaglandins and LB5 might be expected [11,13]. In addition, decreased production of thromboxane A2 may also influence the rejection process as reported previously [27].
Despite all this evidence, we did not find that fish oil treatment had any effect on the incidence of acute rejection during the first post-transplant year, thus confirming other reports [28,29]. Rejection occurred in 44% of the fish-oil-treated patients and in 47% of those taking soy oil (P=NS). The incidence of rejection in our centre over the period studied was 40%, based on described immunosuppression. In contrast, in a previous report, fish-oil-treated patients had a significantly lower cumulative incidence of acute rejections during the first post-operative year. In this study, the in situ expression of cytokines was not explored [14].
Several studies in human volunteers have demonstrated that n-3 fatty acid supplementation reduces the capacity of mononuclear cells to generate TNF, IL-1 and IL-2 [1113]. These substances may suppress the immunological responses in animal models [30]. In this study, however, fish oil therapy did not influence in situ expression of cytokines (TNF-, IL-1ß and IL-2) in patients who did not suffer rejections (Table 3
). At the time of acute rejections, a lower in situ expression of IL-1ß and TNF-
was found in the group receiving fish oil as compared with the control group (Table 4
). This marginal difference in cytokine expression between the two groups did not have an impact on the histological severity of rejection, or on renal function at rejection and after anti-rejection therapy. Inflammatory cells account for >80% of the cells in FNAP samples, with endothelial and tubular cells and occasional glomeruli making up the rest. In theory, differences in the percentage of inflammatory cells present in the FNAPs could affect the outcome of the RT-PCR analysis. In our study, the differences observed in the percentages of parenchymal vs inflammatory cells (12 vs 90%) do not seem to influence the results.
TNF, IL-1 and IL-2 are important pro-inflammatory peptides, and it is possible that the suppressive effect of -3 PUFA may be obscured in the setting of intragraft immune events. The dosage and duration of fish oil treatment in this study was similar to those reported in other studies with either positive [14] or negative [28] results. Since
-3 PUFA are incorporated into plasma phospholipids and cell membranes in a dose-dependent manner [14,31], it is possible that higher doses are required in order to obtain a clinical benefit. However, the fact that the dose of
-3 PUFA used in our study significantly reduced triglyceride levels (203167 vs 164174 mg/dl) suggests that at least a biologically effective level was reached. Finally, polymorphisms that correlate with individual differences in cytokine production have been described for the genes encoding TNF-
and IL-1 [32,33]. Thus, it is possible that the beneficial effect of
-3 PUFA treatment may be limited to individuals with the genotype that confers susceptibility to increase cytokine production. Future studies are needed to clarify this point.
Dietary supplementation with fish oil has been said to improve renal function in several clinical conditions [3436], including renal transplantation [14,15]. This putative effect of fish oil might be mediated by changes in the prostaglandin/thromboxane axis. Eicosapentanoic acid competes with arachidonic acid in the cyclooxygenase pathway, thus limiting the production of thromboxane A2, which has potent vasoconstriction and pro-aggregatory effects [3739]. Other studies, however, have failed to confirm this beneficial effect in renal transplant patients [28,40].
We measured the GFR by [51Cr]EDTA clearance, a method superior to creatinine clearance in the transplantation setting [41]. We were unable to demonstrate any effect of -3 PUFA on overall renal function during follow-up. Our results cannot be explained by differences in the daily doses of cyclosporine or in the cyclosporine trough concentration (Table 2
). It is possible that the higher doses of cyclosporine, warranted early after grafting (during the first 3 months), may interfere with fish-oil-induced changes in the prostaglandin/thromboxane axis. Nevertheless, in stable renal transplant patients, dietary fish oil did not modify the acute cyclosporine-induced changes in renal function [40].
As expected, a greater reduction in triglyceride levels was observed in patients receiving fish oil. A similar effect has previously been observed in patients with isolated hypertriglyceridaemia [42] and in stable renal transplant patients [43,44]. The mechanisms involved in this reduction are incompletely understood, although it has been attributed to a lower rate of very low density lipoprotein synthesis [45]. The presence of triglycerides in cell membranes could explain in part the recently reported lower activity of natural killer cells in healthy subjects receiving this compound [46]. However, whether this effect can modulate the immune response to an allograft remains to be established.
In summary, 3 months of treatment with fish oil after renal transplantation does not influence the acute rejection rate and does not have an impact on the severity of these rejections, graft survival or renal function.
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Acknowledgments |
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Notes |
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References |
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