WO2016184591A1 - Composition for renal protection in renal transplant recipients - Google Patents

Abstract

The present invention relates to a method of administering a composition comprising n-3 PUFAs to a patient who has received, or who is scheduled to receive, a renal transplant. Particularly, the invention provides a method to increase the level of n-3 PUFAs or to correct a deficiency of n-3 PUFAs in the patient's blood. Further, the invention provides a composition for use in the treatment or dietary management of a patient who has received, or who is scheduled to receive, a renal transplant, wherein the composition comprises n-3 PUFAs.

Description

COMPOSITION FOR RENAL PROTECTION IN RENAL TRANSPLANT RECIPIENTS

Field of the invention

The present invention relates to a method of administering a renoprotective composition comprising n- 3 polyunsaturated fatty acids (n-3 PUFAs) to a renal transplant recipient. The invention also provides a method of treating or dietary manage a patient who has received, or who is scheduled to receive, a renal transplant with a composition comprising n-3 PUFAs. Particularly, the invention provides a method to increase the level of n-3 PUFAs or to correct a deficiency of n-3 PUFAs in the patient's blood. Further, the invention provides a renoprotective composition comprising n-3 PUFAs, such as a composition for use in the treatment of a patient who has received, or who is scheduled to receive, a renal transplant, wherein the composition comprises n-3 PUFAs.

Background of the invention

Several studies have reported beneficial cardiovascular effects of marine n-3 polyunsaturated fatty acids. To date, no large studies have investigated the potential, benefits of marine n-3 polyunsaturated fatty acids in recipients of renal transplants.

Dietary factors, like the essential marine n-3 polyunsaturated fatty acids (PUFAs), may exert potent biologic effects and several beneficial metabolic effects of marine n-3 PUFAs have been reported, including lipid modulation, plaque stabilization, reduced blood pressure, less artery calcification, and improved endothelial and myocardial function. Furthermore, anti-inflammatory, antiarrhythmic, antiatherosclerotic, antifibrotic and antithrombotic effects have been reported, which may influence the incidence of cardiovascular disease (CVD) and mortality rates. However, recent prospective cohort studies and randomized, controlled trials (RCTs) have shown mixed results, with only moderate beneficial or even neutral effects on major cardiovascular event and mortality rates.

In patients with end stage renal disease (ESRD), favorable effects of marine n-3 PUFAs on cardiovascular morbidity and mortality have been shown. In patients who are uremic treated with hemodialysis, high intake of marine n-3 PUFAs was associated with a lower risk of sudden cardiac death (SCD), and an RCT found a lower cumulative incidence of myocardial infarction (Ml.) after marine n-3 PUFA supplementation, reference is made to Svensson M, Schmidt EB, Jorgensen KA, Christensen JH; OPACH Study Group: N-3 fatty acids as secondary prevention against cardiovascular events in patients who undergo chronic hemo-dialysis: A randomized, placebo-controlled intervention trial Clin J Am Soc Nephrol 1 : 780-786, 2006, and to Friedman AN, Yu Z, Tabbey R. Denski C. Tamez H, Wenger J. Thadhani R, Li Y, Watkins BA: Inverse relationship between long-chain n-3 fatty acids and risk of sudden cardiac death in patients starting hemodialysis. Kidney Int 83: 1 130- 1135, 2013. Although renal transplantation reduces the risk of CVD in patients with ESRD, CVD remains the leading cause of death in renal transplant recipients (RTRs), as discussed by Israni AK et al, PORT Investigators: Predicting coronary heart disease after kidney transplantation: Patient Outcomes in Renal Transplantation (PORT) Study. Am J Transplant 10:338-3S3, 2010. Whether the beneficial effects of marine n-3 PUFAs for patients with ESRD are applicable to renal transplant recipients is unclear. In some RTCs, there have been reports of improved renal function with marine n- 3 PUFA supplementation, as by van der Heide JJ et al, Effect of dietary fish oil on renal function and rejection in cyclosporine treated recipients of renal transplants. New Engl J Med 329: 769-773, 1993, and by J Wesley Alexander, Influence of long chain polyunsaturated fatty acids and ornithine concentrations on complications after renal transplant. Experimental and Clinical Transplantation vol 6, 2, 2008. In the latter study by Alexander there was supplementation with both omega-3 fatty acids and arginine and the results are not seen as sufficient to conclude that omega-3 fatty acids alone have a beneficial effect on renal transplant recipients. Moreover in the study by Alexander the actual renal function (measured as eGFR) was not commented. Further, results related to patient or graft survival are not provided. Graft rejection could be the result of extensive inflammation related to the body's response to a foreign organ, and not necessarily to the kidney function itself. As such the effect of omega-3 on the actual quality of a functioning kidney is not documented and cannot be concluded from the study by Alexander. Most studies, however, reported no effect. Only small RCTs have assessed the effects of marine n-3 PUFAs in RTRs, with insufficient data to evaluate the effects on mortality. Three separate meta-analyses of RCTs focusing on the effect of marine n-3 PUFA supplementation after renal transplantation have reported a significant reduction in triglyceride levels, a minor reduction of diastolic blood pressure and slightly increased concentrations of HDL cholesterol. However, effects on mortality rates could not be evaluated because of a low number of events (only seven deaths in 846 patients) and short follow-up. Reference is made to Lim AK et al: Fish oil treatment for kidney transplant recipients: A meta-analysis of randomized controlled trials. Transplantation 83: 831 -838, 2007, and to Tatsioni A et al: Effects of Fish oil supplementation on kidney transplantation: A systematic review and meta-analysis of randomized, controlled trials. J Am Soc Nephrol 16: 2462-2470, 2005, and to Bonis PA et al: Effects of omega-3 fatty acids on organ transplantation. Evid Rep Technol Assess (Summ) 1 15: 1 - 1 1 , 2005. The assumption that increased intake of marine n-3 PUFAs significantly reduces the risk of cardiovascular morbidity and mortality has been challenged in recent meta-analyses of RCTs, e.g. Lim AK et al, Transplantation.

5;83(7):83 1 -8, 2007 "Fish oil treatment for kidney transplant recipients: a meta-analysis of randomized controlled trials", concludes that there is insufficient evidence from currently available randomized controlled trials to recommend fish oil therapy to improve renal function, rejection rates, and patient or graft survival. Accordingly, there are divergent results from early and recent studies related to n-3 PUFA treatment of RTRs. A new cohort study in RTRs, by the applicant, has now been performed to assess whether plasma phospholipid levels of marine n-3 PUFAs, which derive from consumption offish, seafood, or marine n-3 PUFA supplements, were associated with overall and cause-specific mortality. Further, it has been assessed whether plasma, phospholipid levels of marine n-3 PUFAs were associated with overall graft loss and graft failure (return to dialysis or renal re -transplantation) in surviving patients (death censored graft loss).

Brief Summary of the invention

From the present cohort study it was concluded that for patients having had a renal transplantation higher plasma phospholipid marine omega-3 polyunsaturated fatty acid (n-3 PUFA) levels were independently associated with better patient survival. A better renal function was found for renal transplant recipients with a higher n-3 polyunsaturated fatty acid level, suggesting renoprotective effects of marine n-3 PUFAs. Mortality rates were lower in patients with high marine n-3

polyunsaturated fatty acid levels compared with low levels for all age categories. There was a lower hazard ratio for death from cardiovascular disease with high levels of marine n-3 polyunsaturated fatty acid and a lower hazard ratio for death from infectious disease with high levels of the marine n- 3 polyunsaturated fatty acid eicosapentaenoic acid (EPA). A clear association between the level of blood plasma phospholipid fatty acids and the probability of survival after renal transplantation has hence been found. Further, it has been found that high levels of plasma marine n-3 polyunsaturated fatty acids were associated with better overall and death censored renal allograft survival. Based on this new knowledge it is found crucial that RTRs should have a sufficient and appropriate level of the relevant n-3 polyunsaturated fatty acids. The invention hence provides a method of administering a patient who has received, or who is scheduled to receive, a renal transplant with a renal protective composition of n-3 PUFAs. Hence, the invention provides a method of treating a patient who has received, or who is scheduled to receive, a renal transplant, the method comprising administering the patient with a composition comprising n-3 PUFAs. More, particularly the invention provides a method of treating a patient who has received, or who is scheduled to receive, a renal transplant, the method comprising administering the patient with a composition comprising n-3 PUFAs, to correct a deficiency of said n-3 PUFAs or to increase the level of n-3 PUFA in the blood, or in various organs, including renal transplants, or in other tissue, including subcutaneous fat.

In another aspect the invention provides a composition comprising n-3 PUFAs as a renoprotective agent, particularly for use as a renoprotective agent for renal transplant recipients. Hence, the invention provides a composition for use in the treatment of a patient who has received, or who is scheduled to receive, a renal transplant, wherein the composition comprises n-3 PUFAs. The composition is administered to the patient to correct a deficiency or increase the level of said n-3 PUFAs in the patient's blood or in various organs, including renal transplants, or in other tissue, including subcutaneous fat. By increasing the level of the relevant n-3 PUFAs in the blood to an appropriate level, the method or composition improves the patient's chance of survival, particularly by reducing the risk of CVD and/or reducing the risk of death from infectious disease. Moreover, marine n-3 PUFA level increment improves renal function and renal graft survival.

Brief description of the drawings

Figure 1 is a Kaplan-Meier survival curve for recipients of renal transplants, showing the proportions of surviving patients grouped according to age and marine n-3 polyunsaturated fatty acid (PUFA) levels in weight percentage of total plasma phospholipid fatty acids. Figure 2 is an estimated survival probability curve in recipients of renal transplants in multivariable- adjusted Cox proportional hazard regression model 2.

Figure 3 A and B visualize the association between marine n-3 PUFA levels and overall graft loss (Panel A, Figure A) and death censored graft loss (Panel B, Figure B).

Detailed description of the preferred embodiments The cohort study reported herein has investigated the effects of omega-3 fatty acids on renal function and cardiovascular risk markers in renal transplant recipients. Further, the study has investigated the effects of omega-3 fatty acids on graft loss. The aim of this study was to assess whether plasma phospholipid levels of marine n-3 PUFAs, which derive from consumption of fish, seafood or marine n-3 PUFA supplements, were associated with overall and cause-specific mortality in RTRs. The major finding of the study was an independent and negative association between plasma phospholipid marine n-3 PUFA levels and overall cardiovascular mortality after renal transplantation. An independent and negative association between plasma marine n-3 PUFA levels and overall and death censored graft loss after renal transplantation was also found. Deaths from infectious disease were strongly associated with low levels of EPA. The much higher number of patients and longer follow-up time in the study, compared with earlier studies, provides a possibility to evaluate associations between marine n-3

PUFA levels and patient and graft survival. A steeper decline in renal function during the first 5 years after transplantation in patients with low levels of marine n-3 PUFAs was found, indicating potential renoprotective effects of marine n-3 PUFAs. In summary, it was found lower overall and

cardiovascular mortality risk after renal transplantation with high plasma phospholipid marine n-3 PUFA levels, indicating that RTRs may benefit from marine n-3 PUFA supplementation. Since three out of five cases of graft loss were caused by patient death, the relationship between n-3 PUFA levels and mortality strongly influences the risk of graft loss. Consistent with this finding, we found a lower risk of overall graft loss with higher n-3 PUFA levels. Future RCTs focusing on the effects of marine n-3 PUFAs on traditional and transplant-specific mortality risk factors with adequate sample sizes, follow-up periods, and dosages of marine n-3 PUFA supplements are warranted in renal

transplantation. Our hypothesis is that the findings from the ongoing clinical trial titled "The Effects of n-3 Polyunsaturated Fatty Acids on Renal and Cardiovascular Risk Markers in Renal Transplant Recipients: a Randomized Double Blinded Placebo Controlled Intervention Study" (ClinicalTrials.gov no. NCTO 1744067) will support the present invention. This study will investigate the effects of omega-3 fatty acids on renal function and cardiovascular risk markers in renal transplant recipients, administering Omacor to the renal transplant recipients.

The invention provides a method of administering a patient who has received, or who is scheduled to receive, a renal transplant with a renoprotective composition comprising n-3 PUFAs. The composition is for improving at least one parameter associated with renal function. Well-functioning kidneys filter out wastes and excess fluids that become part of the urine. When the kidneys are not working well, not enough wastes and fluids are removed, and important hormones are not produced. Glomerular filtration rate (GFR) describes the flow rate of filtered fluid through the kidney. GFR can be measured or estimated based on creatinine levels. Creatinine clearance rate is the volume of blood plasma that is cleared of creatinine per unit time and is a useful measure for approximating the GFR.

In one embodiment, the invention provides a method to increase the plasma phospholipid levels of marine n-3 PUFAs in RTRs. In the study reported in the Examples herein the n-3 polyunsaturated fatty acid (PUFA) level is defined as the sum of the plasma phospholipid eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexacnoic acid (DHA) levels in weight percentage (wt%) of total plasma phospholipid fatty acids. The PUFA level, i.e. the sum of the three PUFAs EPA, DPA and DHA, in addition to the level of these individual fatty acids, in blood samples is measured by gas chromatography. Baseline characteristics of the study participants grouped according to marine n-3 PUFA levels are shown in Table 1 in Example 1 , showing that the PUFA level of the study population is divided into quartiles. The median level of marine n-3 PUFAs in plasma phospholipids was 7.95 wt%. High marine n-3 PUFA levels is defined as >7.95 wt%, whereas low marine n-3 PUFA levels is defined as <7.95 wt%. Quartile 4 includes the group of patients having an n-3 PUFA level above 10.03 wt%. In one embodiment, the invention provides a method as disclosed wherein the level of n-3 PUFAs in the plasma phospholipid in blood of the patient who has received, or who is scheduled to receive, a renal transplant, is increased to >7.95 wt% by administering the patient with a composition comprising n-3 PUFAs. In particular, the level of n-3 PUFAs in the plasma phospholipid in blood of the patient is increased to above about 8 wt% of the total fatty acid level. More preferably, the n-3 PUFA level is increased to > 10.03 wt%, such as to above about 10 wt%. As it has been found that the risk of cardiovascular death is reduced already if the level of n-3 PUFAs in the plasma phospholipid in blood of the patient is increased to above 6.21 wt%, a method increasing the level to this point is also encompassed by the invention. In one embodiment, the invention provides a method of treating a patient who has received, or who is scheduled to receive, a renal transplant, the method comprising administering the patient with a composition comprising n-3 PUFAs, In another embodiment, the invention provides a method of dietary management of a patient who has received, or who is scheduled to receive, a renal transplant, the method comprising administering the patient with a composition comprising n-3 PUFAs. In one embodiment, the invention provides a method for correcting a deficiency or increasing the level of n-3 PUFAs wherein the RTR has a particularly low level of n-3 PUFAs, such as for RTR patients having less than about 6.2 wt% n-3 PUFAs of total plasma phospholipid fatty acids, such as for those having less than 4.0 wt%. Accordingly, the invention provides a method to correct the level of n-3 PUFAs in patients' blood having a deficiency of any of these n-3 PUFAs.

Similar results were found for EPA and DHA alone whereas no association was found between DPA levels and mortality. The risk of death from CVD was markedly lower in patients with high marine n-3 PUFA levels compared with low levels. In particular, death from stroke and SCD showed a strong negative association with marine n-3 PUFA levels. Patients with high levels of EPA were also less likely to die from infectious disease and equally associated with nonsepticemia infectious disease mortality as death from septicemia. A negative association between marine n-3 PUFA levels and death censored graft loss was also found, and high levels of plasma marine n-3 polyunsaturated fatty acids were associated with better renal allograft survival. In subgroup analysis, EPA levels, as opposed to DHA levels, were inversely associated with death censored graft loss. EPA exerts several antiinflammatory effects, of which the dose-dependent competitive inhibition of pro-inflammatory metabolites derived from the n-6 PUFA arachidonic acid is considered important. This may reduce the risk of inflammation and subsequent fibrosis in the renal allograft. Further EPA is also important in dose-dependent competitive inhibition of thromboxanes which regulate vasoconstriction of blood vessels in glomeruli, thereby reducing the risk of ischemia. In our study, wherein the polyunsaturated fatty acid median level was 7.95 wt%, a 1 1 % reduced risk of graft loss was found for every 1.0 wt% increase in n-3 polyunsaturated fatty acid level, showing the importance of increasing or correcting the level of n-3 PUFAs in the patient's blood. No association was found between marine n-3 PUFA levels and death by cancer. Preferably, the invention provides a method to correct a deficiency or increase the level of either of EPA and DHA in the blood of the patient who has received, or who is scheduled to receive, a renal transplant. Alternatively, this may be seen as an increased level of PUFAs in various organs, including renal transplants. E.g. the optimized n-3 PUFA level may be found if taking biopsies of the transplanted organ or from subcutaneous fat. Particularly, the present cohort study indicates that moderate to high levels of EPA are associated with a lower risk of death from infectious disease in renal transplant recipient. Further, it is also anticipated that EPA affects the immune system directly by upregulation of T-cells that are only affected by PPAR activation, hence making the body more tolerant for a transplanted organ and there is hence a less risk for rejection by increasing the EPA level. In one embodiment, the invention provides a method as disclosed wherein EPA is administered as a renal protective agent. Accordingly, the invention provides a method to increase the level of EPA in the blood of the patient who has received, or who is scheduled to receive, a renal transplant. Particularly, this is increased in order to reduce the risk of infectious diseases. The level of EPA in the plasma phospholipid is increased preferably to ≥1 .77 wt%, which was the median level of EPA measured in plasma phospholipids. More preferably, the level of EPA is increased to a level of >2.90 wt%, which was the level measured for the fourth quartile. or to above about 3.0 wt%. In one embodiment, the level of EPA is increased to above about 1.5 wt%, such as to above about 2.0 wt%, above 2,5 wt% or above 3.0 wt%. Similarly, in another embodiment the invention provides a method to increase the level of DHA in the blood of the patient who has received, or who is scheduled to receive, a renal transplant. The level of DHA is increased preferably to >5.08 wt%, which was the median level of DHA measured in plasma phospholipids. More preferably, the level of DHA is increased to a level of > 6.17 wt%, which was the level measured for the fourth quartile. Preferably, the combined level of EPA and DHA in the patient's blood is increased to >6.85 wt% and more preferably >7.95 wt%, such as to above about 8 wt%, or above about 9.0 wt%.

The «Omega-3 Index» has been proposed as a new risk factor for death from CHD. The Omega-3 Index expresses EPA+DHA as % of the total fatty acid content of the red blood cell. Red blood cell fatty acid composition reflects long-term intake of EPA + DHA. In a study by Harris et al (Harris WS and Clemens von Schacky. The Omega-3 Index; a new risk factor for death from coronary heart disease. Preventive medicine 2004;39: 212-220) the "Omega-3 Index" was compared with plasma phospholipid EPA + DHA content in 65 blood samples from healthy subjects who had been taking omega-3 capsules for five months. The samples thus reflected steady state conditions. Correlation coefficient between the "Omega-3 Index" and plasma phospholipid EPA+DHA was 0.91 (P < 0.001 ). This relationship was reflected in the following equation: Omega-3 Index (%) = Plasma PL (%) EPA+DHA X 0.97 + 3.43

The study thus demonstrated that there is a close relationship between plasma phospholipid EPA + DHA and the "Omega-3 Index. Hence, in another embodiment the invention provides a method of treating a patient who has received, or who is scheduled to receive, a renal transplant, the method comprising administering the patient with a composition comprising n-3 PUFAs, to correct a deficiency of said n-3 PUFAs or to increase the level in the blood, wherein the Omega-3 index (%) is increased. Preferably, the Omega-3 Index (%) is increased to above about 1 1 .0, more preferably above 13.0. In one embodiment, the Omega-3 Index (%) is increased to above 9.5, as reduced risk of cardiac death can be seen at this level. In another embodiment, the invention provides a method as disclosed above administering the patient with a composition comprising n-3 PUFAs, to correct an imbalance in the ratio of n-6 to n-3 PUFAs in the blood. A risk of total and cause-specific mortality according to n-6 PUFA to n-3 PUFA ratio has been estimated as shown in Example 1, Table S4. The total omega-6 PUFA level is the sum of linoleic acid, gammalinolenic acid, eicosadienoic acid, dihomogammalinolenic acid, arachidonic acid and adrenic acid. In one embodiment, the invention provides a method wherein the ratio of n-6 to n-3

PUFAs in the plasma phospholipid is corrected to a ratio of less than 4.37, and more preferably to less than 3.3 1. In another embodiment, the ratio of n-6 to n-3 PUFAs in the plasma phospholipid is corrected to a ratio of less than about 4.0 and more preferably less than about 3.0. Particularly, by the method of the invention, a RTR's unfavourable composition of arachidonic acid (ARA) in relation to n-3 PUFAs, or to EPA, is corrected. In one embodiment, the ratio of ARA to n-3 PUFAs in the plasma phospholipids is corrected to less than about 1.0, and preferably to less than about 0.75.

The renoprotective composition comprises n-3 PUFA which preferably originate from marine oil, i.e. fish oil. but may also be derived from algae oil, plant based oil or microbial oil. The PUFAs may be in different forms such as free fatty acids, esters, e.g. C1-C4 alkyl esters, preferably ethyl esters, phospholipids, mono/di/tri-glycerides and salts thereof. In a preferred embodiment, the composition comprises either of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). or a mixture of such, and most preferably these are in the ethyl ester form. The n-3 PUFAs of the composition have been found to have renoprotective effects, and act as renoprotective agents.

In one embodiment, the composition comprises a fatty acid mixture of at least 25 weight % of EPA and/or DHA, more preferably at least 35%. 45%, 55%, 65%, 75%, 85% or 95% of EPA and/or DHA. In one embodiment the composition comprises high concentrations of one fatty acid, preferably either EPA or DHA. For instance, the composition may comprise a fatty acid mixture of at least 80 % EPA, such as at least 90 % EPA, e.g. about 97 % EPA. In one embodiment, the composition comprises at least 55 weight% of at least one of EPA and DHA by the weight of the fatty acids therein. In at least one embodiment, the composition comprises at least 75 weight% of at least one of EPA and DHA by weight of the fatty acid therein, such as at least 80 weight%, such as about 84 weight% of at least one of EPA and DHA by weight of the fatty acids in the composition.

In some embodiments of the present application, the weight ratio of EPA: DHA of the composition ranges from about 1 : 10 to about 10: 1, from about 1 :8 to about 8: 1 , from about 1 :6 to about 6: I , from about 1 :5 to about 5: 1, from about 1 :4 to about 4: 1 , from about 1 :3 to about 3: 1, or from about 1 :2 to 2 about: 1. In at least one embodiment, the weight ratio of EPA.DHA of the composition ranges from about 1 :2 to about 2: 1. In at least one embodiment, the weight ratio of EPA:DHA of the composition ranges from about 1 : 1 to about 2:1. In at least one embodiment, the weight ratio of EPA:DHA of the composition ranges from about 1.2 to about 1.3.

In another embodiment, the composition comprises specific mixtures of EPA and DMA, Such compositions include, but are not limited to the following: about 360 mg EPA and 240 mg DHA pr g oil, 400 mg EPA and 200 mg DHA pr g oil, 500 mg EPA and 200 mg DHA pr g oil, 150 mg EPA and 500 mg DHA. pr g oil, 460 mg EPA and 380 mg DHA pr g oil, above 900 mg EPA pr g oil, above 900 mg DHA pr g oil, and 97% EPA. The total doses are e.g. from 1 -20 g in total of EPA and DHA. In addition, other omega-3 fatty acids than EPA and DHA may be present.

In a preferred embodiment, the composition for use according to the invention comprises an n-3 PUFA mixture of about 84 weight⁰/. EPA and DHA, preferably each capsule comprising 460 mg EPA-ethyl ester and 380 mg DHA-ethyl ester, such as for the pharmaceutical named Omacor, Lovaza or generics of these.

In some embodiments of the present disclosure, the composition acts as an active pharmaceutical ingredient (API), !n some embodiments, the fatty acid of the composition is present in a

pharmaceutically-acceptable amount, the composition is then called a pharmaceutical composition, and is for medical use. As used herein, the term "pharmaceutical ly-effective amount" means an amount sufficient to treat, e.g., reduce and/or alleviate the effects, symptoms, etc., of at least one health problem in a subject in need thereof. In at least some embodiments of the present invention, the composition does not comprise an additional active agent. In this embodiment, the composition may be used in pharmaceutical treatment of patients who has received, or who is scheduled to receive, renal transplants. The composition, use of this and methods using this, has the ability to reduce the risk factors associated with renal transplants. Such risk factors are overall mortality and cause-specific mortality in RTRs, cardiovascular morbidity and mortality, myocardial infarction (MI), sudden cardiac death (SCD), stroke and infectious disease, graft loss, and reduced renal function. The compositions of n-3 polyunsaturated fatty acids may exert beneficial effects on inflammation, fibrosis, endothelial function, lipid profile and blood pressure that may improve renal function and prevent graft loss. Where the composition is a pharmaceutical composition, the composition preferably comprises at least 75 percent of at least one of EPA and DHA by weight of the fatty acids in the composition. For example, in one embodiment, the composition comprises at least 80 percent EPA and DHA, such as at least 85 percent, at least 90 percent, or at least 95 percent, by weight of the fatty acid therein. In another embodiment, the composition according to the invention is a food supplement or a nutritional supplement comprising at least one of EPA and DHA. Further, in one embodiment the composition is a food supplement, a dietary supplement, a nutritional supplement, over-the-counter (OCT) supplement, medical food, or pharmaceutical grade supplement. In a related embodiment, the invention provides a composition selected from the group of Enteral Formulas for Special Medical Use, Foods for Specified Health Uses, Food for Special Medical Purposes (FSMP), Food for Special Dietary Use (FSDU), Medical Nutrition and a Medical Food. Such composition is particularly suited for patients having a deficiency of certain nutrients, such as the n-3 PUFAs. Such composition is typically administered to the subject under medical supervision. In this embodiment, the treatment includes a nutritional treatment. The composition functions as a renoprotective composition. The composition comprises the relevant n-3 PUFAs, particularly EPA and/or DHA, to increase or correct the level of the n-3 PUFAs in the blood of a patient who has received, or who is scheduled to receive, a renal transplant. Accordingly, the composition for use in the treatment or dietary management of a patient who has received, or who is scheduled to receive, a renal transplant, is selected from the above group. In a preferred embodiment, the composition is, or forms part of, Medical Food suitable for administration to RTRs. Also for this food supplement or nutritional supplement it is preferred that the composition is a high concentrated composition of EPA and/or DHA. Preferably, the composition comprises at least 55 \vt% of at least one of EPA and DHA by weight of the fatty acids therein.

The composition and the method of the invention has the ability to correct a nutritional deficiency in a target population. A deficiency means that the patient has a level of n-3 PUFA below the average level or that the target population has a special need. The target population is a group of patients who has received, or who is scheduled to receive, renal transplants. Accordingly, the patient may not have obtained sufficient levels from their diet to cover the increased need related to the existing health condition, or have a particular benefit of high levels of EPA and/or other PUFAs. In one embodiment, the composition is suited for a nutritional or dietary management of renal transplant recipients having a distinctive nutritional requirement. Such composition is typically administered to the subject under medical supervision. By optimizing the level of n-3 PUFAs health benefits may be seen for this target population, reasons being e.g. that n-3 PUFAs may be used more often as substrate in the

prostaglandin biosynthesis, rather than arachidonic acid being used, increased biosynthesis of n-3 PUFA derived tromboxanes, and hence regulation of renal perfusion and for fibroblast activation in renal cell cultures. The invention hence provides nutritional correction of risk factors associated with renal transplants. The risk factors such as overall mortality and cause-specific mortality in RTRs, cardiovascular morbidity and mortality, myocardial infarction (Ml), sudden cardiac death (SCO), stroke and infectious disease can be reduced by using the method or the composition of the invention, independent of the use as a pharmaceutical or as a supplement. Further, the risk of graft loss can be reduced by using the method or the composition of the invention. Accordingly, the present disclosure encompass methods of improving at least one parameter associated with renal function for a subject who has received, or who is scheduled to receive, a renal transplant. The composition may be administered, e.g., in capsule, tablet or any other suitable form for drug or dietary delivery, to the subject for at least one of beneficially affecting: inflammation, fibrosis, endothelial function, lipid profile or blood pressure, and this may accordingly improve, maintain or prevent decline in renal function, prevent graft loss, or reduce the risks associated with such transplantations.

Hence, the invention provides a method and a composition for RTRs wherein the risk of death, cardiovascular morbidity, infectious disease and/or graft loss is reduced. Further, the renal function may be improved, maintained or the decline in renal function may be slowed down. The renal allograft survival may also be improved. Particularly, by the method or use of the invention the renal function can be improved or maintained by reducing, maintaining or preventing an increase of the level of serum creatinine over time. In one embodiment, the invention provides a method of administering a patient who has received, or who is scheduled to receive, a renal transplant with a renal protective composition as disclosed to maintain, stabilize, improve or prevent decline of the function of the transplanted kidney. This is preferably identified as a maintained or improved glomerular filtration rate (GFR), or a slower reduction of this. By the method using the renoprotective composition new damage to the kidney can be prevented, and a worsening of the glomeruli and their filtering properties is prevented. In one embodiment, the method includes a step of estimating the GFR, based on creatinine level, preferably based on a blood test taken. More preferably, the method includes a step of measuring the GFR (mGFR), and not estimating it. Such measurements and estimations should be done regularly, such as e.g. yearly. In one embodiment measured GFR (mGFR) is identified using urinary or plasma clearance of exogenous filtration markers, such as e.g. measuring plasma clearance of iohexol. By the method or use of the compositions according to the invention the following can be prevented: returning to dialysis and/or re-transplant of kidney; or

lowering of GFR above 5 ml/min (absolute value) for a single episode: or

lowering of GFR above 2 ml/min/year (delta value); or

lowering of GFR above 5 ml/min over a life perspective, or preferably lowering GFR less than 10 ml/min, 15 ml/min, 20 ml/min, 25 ml/min, 30 ml/min, 35 ml/min, 40 ml/min, 50 ml/min,

55 ml/min or 60 ml/min over a life perspective; or

lowering of GFR of more than 10% per year.

The reductions in GFR which are prevented are either eGFR or mGFR. Further, by the method or use of the compositions according to the invention the following is prevented: a creatinine increase to above 200 μιτιοΙ/Ι, such as to above 250 μπιοΐ/ΐ or 300 μιηοΐ/l; or - a creatinine increase above 20% for a single episode; or

- a creatinine increase above 10%, such as above 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% - or 100% over a life perspective; or

a creatinine increase above 10 % per year.

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In one embodiment, the invention provides a method as disclosed wherein the GFR is maintained above 10 ml/min, preferably above 20 ml/min, more preferably above 30 ml/min and most preferably above 40 ml/min, or even above 50 ml/min. Alternatively, the method may also include a

measurement of inflammation and/or fibrosis directly in the kidney.

In summary, use of the composition or the method according to the invention provides a reduced risk of overall mortality and cause-specific mortality in RTRs, cardiovascular morbidity and mortality, myocardial infarction (MI), sudden cardiac death (SCO), stroke or infectious disease, or it provides an improved renal function, or it provides beneficial effects on inflammation, fibrosis, endothelial function, lipid profile or blood pressure, or it prevents graft loss. Some groups of renal transplant recipients have been found to have a higher risk of graft loss or death censored graft loss than others, as shown in table 6 of Example 2, and may have a particular advantage of increasing their level of n-3 PUFAs. Hence, in one embodiment of the invention, the patient is a current smoker, has or has had coronary artery disease, has or has had diabetes mellitus, or has experienced an early rejection episode.

In one embodiment, the method of the invention includes a step of measuring the level of n-3 PUFAs in the patient's blood. Such measuring may be done once or several times, and may be done regularly to check whether the levels of PUFAs are at an acceptable level, or to decide on the dose to be administered. The PUFA level may be measured in the plasma phospholipids, e.g. by gas

chromatography, or alternatively the level of PUFAs may be measured in the red blood cells. In one embodiment, the dose of the composition, is adjusted based on the result of the measurement in order to correct the deficiency, increase the level or correct an imbalance of omega-6:omega-3 ratio. In one embodiment, it may be decided to switch to another renoprotective composition, e.g. having a higher concentration of certain PUFAs, such as to a higher concentration of EPA. In one embodiment, the method includes steps wherein both the n-3 PUFA levels and the creatinine levels, and/or GFR, are measured, and the method of treatment or dietary management, including e.g. the dose, is adjusted according to the results of these. If e.g. the level of omega-3 PUFAs is below the recommendation, e.g. below 8 wt% or 10 wr% of the total fatty acid level, or if the creatinine level is too high, such as above 200 pmol/l, the total dose of omega-3 may be increased. The total daily dosage of the composition may range from about 0,600 g to about 6..000 g. For example, in some embodiments, the total dosage of the composition ranges from about 0,800 g to about 4.000 g, from about 1.000 g to about 4.000 g, such as 3.000 g, or from about 1.000 g to about

2.000 g. For instance, a daily dose of about 2.0 grams of Omacor over time is likely to be appropriate for most patients to achieve the threshold value of about 10 wt% n-3 PUFAs of the total plasma phospholipid level.

The composition may be administered in from 1 to 10 dosages, such as from I to 4 times a day, such as once, twice, three times, or four times per day, and further for example, once, twice or three times per day. The administration may be oral or any other fonn of administration that provides a dosage of n-3 PUFAs to a subject. In a preferred embodiment, the subject is administered with capsules of 1 g three times a day, preferably wherein the capsules each comprise 460 mg EPA-ethyl ester and 380 mg DHA-ethyl ester. In one embodiment, the dose is adjusted according to the level of n-3 PUFAs measured for the individual patient.

The compositions may further comprise at least one antioxidant. Examples of antioxidants suitable for the present disclosure include, but are not limited to, a-tocopherol (vitamin E), calcium disodium EDTA, alpha tocoferyl acetates, butylhydroxytoluenes (BHT), butylhydroxyan isoles (BHA) and ascorbyl palmitate.

The compositions presently disclosed may be administered, e.g., in capsule, tablet or any other drug or dietary delivery forms. For example, the composition may be encapsulated, such as in a gelatin. Formulated forms of all the above are all encompassed by the definition of the composition. Examples of such formulations are Self Micro Emulsifying Drug Delivery System (SMEDDS), Self Nano Emulsifying Drug Delivery Systems (SNEDDs) and Self- Emulsifying Drug Delivery Systems (SEDDs) which form an emulsion in an aqueous solution. For example, the composition may be in the form of a pre-concentrate of any of the above which spontaneously form an emulsion when mixed with gastric/intestinal fluid. Such emulsions, when formed, may provide for increased or improved stability of the fatty acids for uptake in the body and/or provide increased surface area for absorption. Further, the composition may be in the form of emulsions and formulations where the

active/nutritional ingredient is microencapsulated or in the form of a gel or semi-solid formulations.

The composition is administered to patients who has received, or who is scheduled to receive, renal transplants. In one embodiment, the treatment, either pharmaceutical or nutritional, should start well before the transplantation, such as e.g. 0-2 weeks prior to surgery, e.g. 0-4 weeks prior to surgery, or e.g. 0-6 weeks, or 0-8 weeks prior to surgery. If possible, based on the condition of the patient, it may be advisable to start increasing the level of the n-3 PUFAs prior to transplantation. In the cohort study disclosed herein, n-3 PUFA levels in plasma phospholipids in blood were measured by gas chromatography 10 weeks after transplantation. Based on the association found between higher plasma phospholipid marine n-3 PUFAs measured at this stage with better patient survival, the treatment should start soon after transplantation, if it has not started before, such as 0- 12 weeks after transplantation, preferably 0- 10 weeks after transplantation, such as 0-8 weeks after the

transplantation. In one embodiment, it may be advisable to wait to start the therapy to some weeks post-surgery when the conditions have normalized. The composition is preferably administered over a long period, such as 12-52 weeks, e.g. 24-46 weeks. In one embodiment, the patient should continue to take the composition for the rest of the life. In a second aspect the invention provides a renoprotective composition comprising n-3 PUFAs, particularly for use as a renoprotective composition for renal transplant recipients. Hence, the invention provides a composition for use in the treatment or in dietary management of a patient who has received, or who is scheduled to receive, a renal transplant, wherein the composition comprises n- 3 PUFAs. The composition is administered to the patient to correct a deficiency or increase the level of said n-3 PUFAs in the patient's blood. In one embodiment, the invention provides non-medical use of the renoprotective composition as disclosed, as a supplement as described above. Similarly, the invention provides a composition of n-3 PUFAs as disclosed for the treatment of renal transplant recipients, such as for treatment of at least one health problem associated with renal transplantations. This aspect include the same embodiments as outlined above for the first aspect. Preferred embodiments of the invention will now be described by way of example only and with reference to the accompanying Figures, in which Figure 1 is a Kaplan-Meier survival curve for recipients of renal transplants, showing the proportions of surviving patients grouped according to age (age <60 and >60 years old) and marine n-3 polyunsaturated fatty acid (PUFA) levels (the sum of eicosapentaenoic, docosapentaenoic, and docosahexaenoic acid levels in weight percentage [wt%] of total plasma phospholipid fatty acids <7.95 wt% [low] and >7.95 wt% [high}.

Figure 2 is an estimated survival probability curve in recipients of renal transplants in multivariable- adjusted Cox proportional hazard regression model 2. Shown are the survival probabilities of patients belonging to quartifes 1 - 4 according to marine n-3 polyunsaturated fatty acid (PUFA) levels (the sum of eicosapentaenoic, docosapentaenoic, and docosahexaenoic acid levels in weight percentage (wt%) of total plasma phospholipid fatty acids after adjustment for the following variables: recipient age, sex, n-6 PUFA levels, eGFR using the Modification of Diet in Renal Disease formula, time in dialysis before transplantation, preemptive transplantation, body mass index, number of antihypertensive drugs, diabetes mellitus, coronary artery, cerebrovascular and peripheral vascular diseases, and albumin and total plasma cholesterol concentrations. Quartile 1, n-3 PUFA <6.20 wt%; quartile 2, n-3 PUFA =6.21-7.94 \vt%; quartile 3, n-3 PUFA =7.95-10.02 wt%; quartile 4, n-3 PUFA > 10.03 wt%.

Figure 3 provides the association between marine n-3 PUFA levels and overall graft loss (Panel A, Figure A) and death censored graft loss (Panel B, Figure B) adjusted for recipient age and n-6 PUFA levels in marine n-3 polyunsaturated fatty acid (PUFA) spline models. The log hazard of both overall and death censored graft loss decreased linearly with higher levels of marine n-3 PUFAs. The dots represent the presence of marine n-3 PUFA measurements, the central dotted line represents the point estimates and the gray shade represents the 95% confidence interval.

Examples:

Example 1: Observational cohort study of recipients of renal transplants and the association between marine n-3 polyunsaturated fatty acid levels and mortality

Study Design and Population In Norway, all organ transplantations are performed at Oslo University Hospital, Rikshospitalet. From a population of approximately 5 million inhabitants, 2978 renal transplantations were performed in 2837 patients with ESRD between September 30, 1999 and October 13, 201 1. Patients under the age of 16 years old (n=78) and patients who were transferred to their local hospitals (n=335), suffered graft loss (n=58), or died (n=21 ) within the first 10 weeks post-transplant were not eligible for participation in the study. Most patients attended our outpatient clinic for the first 10 weeks after renal transplantation. Informed consent was obtained from 2001 of 2345 eligible patients, in whom blood samples were drawn, measurements were taken, and clinical information was recorded at a final appointment 10 weeks post-transplant. From eligible patients, 344 patients did not attend the final appointment because of reduced capacity at our laboratory during 2007 and 2008. They were not included in the study because of the lack of laboratory and clinical data. In 1 1 patients, the amount of plasma was too small for individual fatty acids to be adequately analyzed. The remaining 1990 patients were included in this study.

Standard treatment protocol

All R^'I^'R received a combination of prednisolone, calcineurin inhibitors and cell proliferation inhibitors. Up to 2007 all patients were treated with cyclosporine A. From 2007 patients under the age of 50 years with a normal oral glucose tolerance test and a body mass index < 28 kg/m² were treated with tacrolimus instead of cyclosporine A. From 2009 steroid doses were reduced. Prior to

2001 the patients were treated with azathioprine, after which it was substituted with mycophenolate.

In addition, in the year 2000 and after 2007, induction therapy with basiliximab was given.

Rejections were treated with intravenous methylprednisolone followed by an increased dose of oral prednisolone. Steroid-resistant rejections were treated with anti-thymocyte globulin or anti-CD3 monoclonal antibodies.

Data Collection and Registry

In study participant blood samples were drawn in the fasting state at the clinical appointment 10 weeks post-transplant. Some samples were analyzed at a central biochemical department, and laboratory test results were entered uniformly into a database. Other blood samples were immediately frozen, stored at -80°C, and later on sent to The Lipid Research Center, Aalborg University Hospital for analysis of plasma phospholipid fatty acid composition by gas chromatography.

End point data were collected from The Norwegian Renal Registry, The registry is on the basts of annual reports from all nephrologists working in Norway and includes all patients on renal replacement therapy (RRT, i.e. dialysis or renal transplantation). Mortality is continuously registered. Surviving patients were censored on February 1 , 2014. Mortality end points were defined according to the European Renal Association -European Dialysis and Transplant

Association causes of death and included the broad categories cancer, infectious disease, and cardiovascular mortality (from the latter category, death from stroke, myocardial infarction (MI], and sudden cardiac death (SCD)). In a random selection of 200 patients, all variables included in the Cox models were checked for consistency between data continuously registered at The

Norwegian Renal Registry and data retrieved from medical records. There was excellent correspondence of data, with the exception of smoking status. After reviewing the medical records, correcting smoking status data, and obtaining missing data of all variables in 1990 study participants, there was 0.6% missing data on smoking status and <0.5% for other variables, and for most variables, there were no missing data. In total, there were only 14 study participants (0.7%) with missing data. They were not included in the multivariablc Cox regression analysis. Baseline characteristics of patients with missing data did not differ significantly from patients without missing data. In short, individual fatty acids were identified by gas chromatography and quantitated as the weight-percentage (wt %.) of total fatty acids. In this study, marine n-3 PUFAs were defined as the sum of plasma phospholipid levels of three individual marine n-3 PUFAs: eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA).

Extraction of lipids

Extraction of total lipids was performed by a modified Folch method, see Folch J, Lees M, Sloane Stanley GH: A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem, 1957; 226( 1 ):497-509. Five hundred uL plasma was extracted with 5 mL of chloroform- methanol (2: 1 ) containing 50 ug/mL butylated hydroxytoluene (BHT) as antioxidant. After adding 0.75 mL of 0.9% sodium chloride, the tubes were mixed and centrifuged at 3220 g at 10° C for 10 minutes. The upper aqueous phase was discharged and the protein disk reextracted with 5 mL of chloroform-methanol (2: 1 ) containing 50 ug/mL BUT and 1 mL 0.9% sodium chloride. The organic phases were combined and dried under nitrogen for 45 minutes at 40° C and dissolved in 1 mL of chloroform. Separation and analysis of plasma phospholipid fatty acids

The phospholipid fraction was isolated essentially as described by Burdge, see Burdge GC, Wright P, Jones AE, Wootton SA; A method for separation of phosphatidylcholine, triacylglycerol, non- esterified fatty acids and cholesterol esters from plasma by solid-phase extraction. Br J Nutr.

2000;84(5):78 I -7. The lipids dissolved in 1 mL chloroform were transferred to a Bond Elut NH2 column 200mg. Agilent Technologies, US) preconditioned with 4 mL of hexane followed by washing with 4 mL of chloroform. The phospholipid fraction was e luted with 2 mL chloroform- methanol (3:2) followed by 2 mL of methanol, after wh ich the phosphol ipid fraction was dried under nitrogen for I hour at 40° C,

Transmethylation of phospholipid fatty acids was performed after dissolving in 500 uL warm heptane (50° C), mixing briefly and then adding 25 uL of 2M potassium hydroxide in methanol and heating for 2 minutes at 50° C. After mixing, the tubes were centrifuged at 3220 g for 10 minutes at 10° C and the upper phase transferred to gas chromatographic injection tubes.

The fatty acids were quantitated using a Varian 3900 gas chromatograph with a CP-8400

autosampler, a flame ionization detector and a CP-Sil 88 60 m x 0.25 mm capillary column (Varian,

Middleburg, The Netherlands). We used a split injection mode, constant flow rate, temperature programing from 90 - 210° C and helium as the carrier gas. Fatty acids were identified from their relative retention time, and quantitated as the weight percent of total fatty acids (wt%).

Results

Baseline characteristics of the study participants grouped according to marine n-3 PUFA levels are shown in Table 1. The median level of marine n-3 PUFAs in plasma phospholipids was 7.95 wt%. Patients with high marine n-3 PUFA levels (>7.95 wt%) were older than patients with lower levels (<7.95 wt%). From 2007, most patients under the age of 50 years old were treated with tacrolimus, whereas older patients received cyclosporin A. When adjusted for age and transplant era, neither choice of calcineurin inhibitor nor eGFR differed between high and low levels of marine n-3 PUFAs, We found a negative association between marine n-3 PUFA levels and both current smoking and n-6 PUFA levels, even after adjustments for age and transplant era, and a trend toward less living donor transplantations and lower prevalence of diabetes mellitus w ith high marine n-3 PUFA levels. Adult RTRs not included in the study were older (mean age of 55.1 years old) than the study participants (mean age of 51.6 years old). Because there were more participants not included in the study after 2007, the groups differed with regards to choice of calcineurin inhibitors (Table S3). When adjusting for age, other baseline characteristics for the two groups were similar.

During the study period, the total number of deaths was 406 (20.4%). In 164 patients, death was caused by CVD (40.4% of deaths). There were 95 deaths by cancer (23.4%) and 101 deaths by infectious disease (24.9%). The median follow-up time for study participants was 6.8 years. The crude analysis showed a small positive association between levels of marine n-3 PUFAs and mortality. However, in an age-stratified analysis reducing the con-founding effect of recipient age, the mortality rate was lower in participants with marine n-3 PUFA levels at or above the median value of 7.95 wt% compared with the patients with lower marine n-3 PUFA levels for all age categories (Figure 1 , Table

2) . The pooled estimate for the mortality ratio was 0.69 (95% confidence interval [95% CI), 0.57 to 0.85). Consistent with this finding, we found a negative association between marine n-3 PUFA levels and mortality in multivariable-adjusted Cox proportional hazard regression analysis using both models 1 and 2. The association between marine n-3 PUFA levels and mortality was similar in periods with high and low inclusion rates.

We found lower levels of n-6 PUFAs with higher levels of marine n-3 PUFAs (Table S3).There was no interaction between marine n-3 PUFA and n-6 PUFA levels. The mean ratio of n-6 PUFA to n-3 PUFA was 4.75. When using marine n-6 PUFA to n-3 PUFA ratio (Table S4) or AA to EPA ratio as the primary exposure, we found similar results to using marine n-3 PUFA levels alone. Although there were differences in hazard ratio (HR) estimates between models 1 and 2, similar trends were found for all causes of death with both models (Table 3). When grouped according to marine n-3 PUFA levels comparing the upper with the lower quartile, there was a 56% lower risk of death (multivariable-adjusted HR, 0.44; 95% 0,0.26 to 0.75) using model 1 (Table 3) and 67% lower risk of death (multivariable-adjusted HR, 0.33; 95% CK 1.19 to 0.58) using model 2 (Figure 2, Table 3). Similar results were found for EPA and DHA alone whereas no association was found between DPA levels and mortality. The risk of death from CVD was markedly lower in patients with high marine-3 PUFA levels compared with low levels. In particular, death from stroke and SCD showed a strong negative association with marine n-3 PUFA levels (Table

3) . Patients with high levels of EPA were also less likely to die from infectious disease and equally associated with nonsepticemia infectious disease mortality as death from septicemia (Table S I). No association was found between marine n-3 PUFA levels and death by cancer. In 908 of 984 patients (92.2%) with functional renal grafts 5 years post-transplant, we looked at the decline in renal function over time according to marine n-3 PUFA levels. Creatinine values increased more in patients with low marine n-3 PUFA levels than in patients with higher marine n-3 PUFA levels (Table 4).

Table 1: Baseline characteristics of the study participants according to levels of marine n-3 polyunsaturated fatty acids.

Table 1 provides the baseline characteristics or the study population according to marine n-3 polyunsaturated fatty acid (PUFA) levels defined as the sum of plasma phospholipid eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid levels in weight percentage (wt%) of total plasma phospholipid fatty acids. Results are presented as proportions for categorical data, medians and interquartile ranges for time in dialysis, and means and SDs for other continuous data. Differences in baseline characteristics were evaluated using the Mantel-Haenszel test of linear trend for categorical data, the Kruskal- Wallis test fortime in dialysis, and linear regression for other continuous data. Pretransplant diabetes mellitus, coronary artery, and cerebrovascular and peripheral vascular disease were recorded before first renal transplantation. Recipient and donor age, deceased or living donor, time in dialysis, number of HLA mismatches, and smoking status were recorded at the time of transplantation. Choice of calcineurin inhibitor, number of antihypertensive drugs, delayed graft function, early rejection episodes, body mass index, eGFR using the Modification of Diet in Renal Disease formula, total cholesterol, and albumin values were recorded at a clinical appointment 10 weeks post-transplant.

Table 2. Mortality rates by marine n-3 polyunsaturated fatty acid level and age category

Table 2 provides the mortality rate according to marine n-3 polyunsaturated fatty acid (PUFA) levels (the sum of eicosapentaenoic, docosapentaenoic, and docosahexaenoic acid levels in weight percentage (wt%) of total plasma phospholipid fatty acids; >7.95 wt% [high) and <7.95 wt% (low)) in different age groups. Mortality rate ratios for each age category were obtained by dividing the mortality rate of patients with high marine n-3 PUFA levels by the mortality rate of patients with low levels. Table 3: Estimated mortality risk according to quartiles of marine n-3 polyunsaturated fatty acid levels using multivariable Cox proportional hazard regression analysis.

Table 3 provides the estimated risk. of total and cause-specific mortality using multivariable-adjusted Cox proportional hazard regression models 1 and 2. Results are presented as multivariable-adjusted hazard ratios for developing mortality end points relative to the lower quartile of marine n-3 polyunsaturated fatty acid (the sum of eicosapentaenoic acid, docosapentaenoic acid, and

docosahexaenoic acid in weight percentage (wt%) of total plasma phospholipid fatty acids) levels, in addition to marine n-3 polyunsaturated fatty acids levels, model 1 included the following variables: recipient age, a product term of recipient age and marine n-3 polyunsaturated fatty acid levels, sex, eGFR using the Modification of Diet in Renal Disease formula, time in dialysis before transplantation, preemptive transplantation, body mass index, number of antihypertensive drugs, diabetes mellitus, coronary artery, cerebrovascular and peripheral vascular disease, and albumin and total plasma cholesterol concentrations. Model 2 also includes n-6 polyunsaturated fatty acids levels as a covariate. HR, hazard ratio; 95% CI, 95% confidence interval; Ml, myocardial infarction; SCD, sudden cardiac death.

Tabic 4. Change in renal function within the first 5 years after transplantation according to levels of marine n-3 polyunsaturated fatty acids.

Table 4 provides the change in renal function in the first 5 years post-transplant in study participants with functional renal grafts transplanted between 1999 and 2008 according to marine n-3

polyunsaturated fatty acid (PUFA) levels defined as the sum of plasma phospholipid eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid levels in weight percentage (wt%) of total plasma phospholipid fatty acids. Results are presented as means and SDs for serum creatinine values. Statistic trend was evaluated by linear regression analysis.

Statistical Analyses

Differences in baseline characteristics by quartiles of marine n-3 PUFAs according to plasma phospholipid levels were evaluated using the Mantel-Haenszel test of linear trend for categorical data, the nonparametric Kruskal-Wallis test for time in dialysis, and linear regression for other continuous data. The associations between total and individual marine n-3 PUFAs and mortality end points were studied by stratified and Cox proportional hazard regression analyses. A stratified analysis adjusting for recipient age was used for estimation of mortality rates. Identification of confounders and fitting of Cox models and graphing are described below. In short, recipient age was identified as a strong confounder and an effect-measure modifier for marine n-3 PUFAs; hence, a product term was included in the survival analysis. Two Cox models were fitted, in addition to recipient age and a product term of recipient age and marine n-3 PUFA level, model 1 included the following traditional and transplant-specific mortality risk factors: sex, eGFR according to the Modification of Diet in Renal Disease fonnula, time in dialysis before transplantation, preemptive transplantation (no dialysis treatment before transplantation), choice of calcineurin inhibitor (treatments with cyclosporine A or tacrolimus were included as two separate categorical variables), smoking status (current smoker, former smoker, or lifelong nonsmoker as a categorical variables), body mass index, albumin and total plasma cholesterol concentrations, number of antihypertensive drugs, diabetes mellitus before transplantation, pretransplant coronary artery, and cerebrovascular and peripheral vascular disease. Model 2 added n-6 PUFA levels as a co-variate. A review of the rationale for the two models is found below. Model assumptions were checked by inspection of the log-log survival time plots and formal hypothesis tests (Schoenfeld residuals). A two-sided P value of <0.05 was considered statistically significant. PASW Statistics,version 17.0 (IBM, New York, NY) and STATA, version 13.0 (Stata Corp, College Station, TX) were used for the statistical analysis.

Identification of confounders and effect-measure modifiers, fitting of Cox proportional hazard models and graphing Stratified analysis was used to identify mortality risk factors that confounded the effects of marine n- 3 PUFA. Recipient age was identified as a strong confounder and mortality rates by marine n-3 PUFA was studied in different age categories. Additional confounding was introduced by eGFR, albumin and n-6 PUFA levels (the sum of arachidonic, linoleic, gammalinoleic, eicosadienoic, dihomogammalinolenic and adrenic acid levels). Other traditional and transplant-specific mortality risk factors in RTR, as described in Israni AK, Snyder JJ, Skeans MA, Peng Y, Maclean JR,

Weinhandl ED, Kasiske BL; PORT Investigators: Predicting coronary heart disease after kidney transplantation: Patient Outcomes in Renal Transplantation (PORT) Study. Am J Transplant.

2010(2):338-53, were included as predefined variables in both Cox models. Recipient age was identified as an effect-measure modifier for marine n-3 PUFA and a product term was included in the Cox models. We identified no other two-way interactions that affected the result or any collinear variables among the covariates in the Cox models.

Since we do not have any dietary data to adjust for effects of various nutrients, we cannot know whether dietary intake of marine n-3 PUFA and n-6 PUFA are individual risk factors or separate or shared risk markers reflecting adherence to a specific diet profile. To rule out potential collider effects, we developed two Cox models, where model 1 excluded and model 2 included n-6 PUFA levels. The rationale for presenting results obtained by both models is that model 1 produced a more conservative estimate of marine n-3 PUFA effects and model 2 included all confounders identified in the stratified analyses and adjusted for the potential influence of n-6 PUFA levels on the relationship between marine n-3 PUFA levels and mortality.

EPA competes with the pro-inflammatory n-6 PUFA arachidonic acid as substrate in the

cyclooxygenase pathway, which produces the prostaglandin hormones that initiate the inflammation process. High levels of EPA may reduce inflammation and subsequent risk of death from infectious disease, cancer or cardiovascular disease, but not independently of levels of arachidonic acid or total n-6 PUFA. The high n-6 PUFA to n-3 PUFA ratio, including both marine n-3 PUFA and alpha- linolenic acid levels, found in populations with a typical Western diet, have been associated with increased risk of cardiovascular morbidity and mortality. In this population, with more patients adherent to a Nordic diet, n-6 PUFA to n-3 PUFA ratios were lower. The higher variance in n-3 PUFA levels compared with n-6 PUFA levels implies that marine n-3 PUFA levels determine most of the variance in n-6 PUFA to n-3 PUFA ratio. This may partly explain why results for n-6 PUFA to n-3 PUFA ratio mirrors that of marine n-3 PUFA levels (Table S4).

Individual marine fatty acids are found in roughly the same mutual proportion in different species of fish and most marine n-3 PUFA supplements, as described in Michas G, Micha R, Zampelas A: Dietary fats and cardiovascular disease: Putting together the pieces of a complicated puzzle.

Atherosclerosis. 2014;234(2):320-8. Therefore, to evaluate the effect of the highly correlated individual marine n-3 PUFA, they were analyzed separately by Cox regression analysis. The imm unosuppress i ve treatment changed over time. We performed age-stratified analysis and Cox regression analysis in three strata according to transplant period (30th of September 1999 to 31 st of December 2006, 1st of January 2007 to 31st of December 2008 and 1st of January 2009 to 13th of October 201 1 ) to adjust for transplant era effects.

Adjusted survival probabilities and corresponding survival probability curves were created using R version 3.0.1 (R Foundation for Statistical Computing, Vienna, Austria). First, we estimated the survival probabilities for each individual from the Cox proportional hazard model 2 with potential con founders averaging over all included patients. The adjusted survival probabilities, time since transplantation and codes for quartiles of covariates were extracted from the fitted model to a new data He for graphing.

Baseline characteristics of patients not included in the study and assessment of bias

Baseline characteristics of adult patients (> 16 years) not included and study participants are described in Table SI . Adult patients not included in the study were older than the study participants. When stratifying for age categories, study participants more often had a living donor, otherwise there were no significant differences between the two groups. The proportion of patients who died during follow-up was lower in adult patients not included in the study ( 11.2% vs 20.4%). However, the study participants were followed for a longer period of time and the overall mortality rate was slightly higher in adult patients not included in the study (mortality rate ratio 1.08). When grouped according to transplant era, the mortality rate for patients included and not included in the study was similar in the era of 2007 to 2008 where many eligible patients had missing blood samples (Supplemental Table S6). .In addition, revision of medical records of 100 non-eligible patients who were transferred early to local hospital revealed a myriad of reasons for transferal not necessarily associated with increased comorbidity or mortality risk. This could provide some explanation to why there were only minor differences in baseline characteristics between study participants and adult patients not included in the study.

Before 2007 and after 2009 the mortality rate was lower in the study participant group (Table S6). However, they were also younger, which would probably influence mortality rates. More importantly, we do not know the composition of plasma fatty acids or have any dietary data for patients not included in the study. Therefore, we do not know if our findings apply to the whole population of adult Norwegian RTR. Nonetheless, when grouping the study participants according to marine n-3 PUFA levels, there were only minor differences in mortality rate ratios for each transplant era despite variance in inclusion rates (Table S2), indicating a limited degree of selection bias in this study. Competing risk, relative survival rates and diagnosis misclassification

We analyzed the effect of marine n-3 PUFA levels on cause-specific mortality in isolation, without adjustments for competing risk. The simple rate ratio interpretations obtained by Cox regression analysis is easy to interpret in contrast to models that adjust for competing risk and regarding death from cancer, we found no associations with marine n-3 PUFA levels. Since cancer and CVD share some common risk factors, patients with an increased risk of death from cancer were also at increased risk of death from CVD. In patients under the age of 45 years, where competing risk from CVD mortality is minimal, patients with high marine n-3 PUFA levels were less likely to die from cancer compared with patients with low levels (mortality rate ratio 0.90). In the higher age groups, patients with high compared with low levels of marine n-3 PUFA were more likely to die from cancer (Table 2), possibly due to the effects of competing risk. However, even with analysis of excess hazard rate for cancer using maximum likelihood estimation of relative survival rate, there was no significant association between marine n-3 PUFA levels and cancer mortality. We found no association between death from Ml and marine n-3 PUFA levels. However, many cases of SCD could be related to Ml and associations between marine n-3 PUFA levels and death from MI would depend on diagnosis classification. In addition, SCD, Ml and stroke mortality rates could be influenced by competing risk, while estimates for overall cardiovascular mortality are probably more reliable.

The supplemental Tables S1-S6 referred to above are found below:

Table SI. Baseline characteristics of adult patients not included in the study compared with the study participants

The table S I provides the baseline characteristics of study participants and adult patients (> 16 years) not included in the study. Results are presented as proportions for categorical data, median and interquartile range for time in dialysis and mean and standard deviations for other continuous data. Differences in baseline characteristics were evaluated using Chi-square for categorical data, Mann-Whitney U-test for time in dialysis and t-test for other continuous data. Pre-transplant diabetes mellitus, coronary, cerebrovascular and peripheral vascular disease were recorded before first renal transplantation. Recipient and donor age, deceased or living donor, time in dialysis and smoking status were recorded at the time of transplantation. Choice of calcineurin inhibitor were recorded at a clinical appointment 10 weeks post-transplant for study participants and within the first weeks after transplantation, for patients not included in the study.

Table S2. The transplantation era effect on mortality rates by marine n-3 polyunsaturated fatt acid levels in different age categories

The table S2 provides the mortality rate according to marine n-3 polyunsaturated fatty acid (PUFA) levels (the sum of eicosapentaenoic, docosapentaenoic and docosahexaenoic acid levels in weight percentage (wt%) of total plasma phospholipid fatty acids; > 7.95 wt% (high) and < 7,95 wt% (!ow) in different transplantation eras. Shown are mortality' rates in patients younger than 60 years and patients aged 60 years or more. Mortality rate ratios for each time period (30th of September 1999 to 31 st of December 2006, 1 st of January 2007 to 31st of December 2008 and 1 st of January 2009 to 13th of October 201 1 ) was obtained by dividing the mortality rate of patients with high marine n-3 PUFA levels by the mortality rate of patients with low levels for each age group.

Table S3. Baseline fatty acid composition of the study participants according to levels of marine n-3 polyunsaturated fatty acids. P for trend.

The table S3 provides the baseline plasma phospholipid fatty acid composition. Patients divided into quartiles according to marine n-3 polyunsaturated fatty acids (PUFA) levels, defined as the sum of plasma phospholipid eicosapentaenoic, docosapentaenoic and docosahexaenoic acid levels in weight percentage (wt%) of total plasma phospholipid fatty acids. Results are presented as means and standard deviations. Trend was evaluated using linear regression. MUFA:

Monounsaturated fatty acids. Q: quartiles.

Table S4. Estimated mortality risk according to quartiles of n-6 to n-3

polyunsaturated fatty acid ratio using multivariable Cox proportional hazard

regression

The table S4 provides the estimated risk of total and cause-specific mortality according to n-6 PUFA to n-3 PUFA ratio using multivariable adjusted Cox proportional hazard regression analysis. Results are presented as multivariable adjusted hazard ratio (HR) for developing mortality endpoints relative to the lower quartile of n-6 to n-3 PUFA ratio (n-6: the sum of linoleic acid, gamma! inolenic acid, eicosadienoic acid, dihomogammalinolenic acid, arachidonic acid and adrenic acid levels in weight percentage (wt%) of total plasma phospholipid fatty acids, n-3 : the sum of alpha-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid levels in weight percentage (wt%) of total plasma phospholipid fatty acids). In addition to n- 6: n-3 ratio, the following variables were included in the model: Recipient age, a product term of recipient age and n-6 to n-3 PUFA ratio, gender, estimated glomerular filtration rate using the Modification of Diet in Renal Disease fonnula, time in dialysis prior to transplantation, preemptive transplantation, body mass index, number of antihypertensive drugs, diabetes mellitus, coronary artery, cerebrovascular and peripheral vascular disease, albumin and total plasma cholesterol concentrations. Q: quartile. CI: confidence interval. Ml: myocardial infarction. SCD: sudden cardiac death.

The table S5 provides the estimated risk of total and cause-specific mortality using multivariable adjusted Cox proportional hazard regression models 1 and 2. Results presented as multivariable adjusted hazard ratio (HR) for developing mortality endpoints relative to the lower quartile of the individual marine n-3 polyunsaturated fatty acids eieosapentaenoic acid and docosahexaenoic acid in weight percentage (wt%) of total plasma phospholipid fatty acids. In addition to either eieosapentaenoic acid or docosahexaenoic acid levels, model 1 included the following variables: Recipient age, a product term of recipient age and either eieosapentaenoic acid or

docosahexaenoic acid as appropriate, gender, estimated glomerular filtration rate using the Modification of Diet in Renal Disease formula, time in dialysis prior to transplantation, preemptive transplantation, body mass index, number of antihypertensive drugs, diabetes mellitus, coronary artery, cerebrovascular and peripheral vascular disease, albumin and total plasma cholesterol concentrations. Model 2 include in addition n-6 polyunsaturated fatty acids levels as a covariate. Q; quartiles. CI: confidence interval. Table S6. The transplantation era effect on mortality rates by study participation

The table S6 provides the mortality rate for adult patients (> 16 years) who were not included in the study and study participants grouped according to transplantation era (30th of September 1999 to 3 1 st of December 2006, 1 st of January 2007 to 31st of December 2008 and 1 st of January 2009 to 13th of October 201 1 ). Mortality rate ratios for each time period was obtained by dividing the mortality rate of adult patients not included in the study by the mortality rate of patients included in the study. Shown are also mean recipient age at the time of transplantation and the proportion of patients participating in the study for each time period. The proportion of participating patients was obtained by dividing the number of patients who received a renal transplant and were included in the study by the sum of all patients who received a renal transplant for each time period.

Example 2: Observational cohort study of recipients of renal transplants and the association between marine n-3 polyunsaturated fatty acid levels and renal allograft survival

As part of the same observational cohort study described in Example 1 associations between plasma marine n-3 polyunsaturated fatty acid levels and graft loss were assessed by multivariable Cox proportional hazard regression analysis. Plasma phospholipid fatty acid composition was determined by gas chromatography and individual fatty acids recorded as weight percentage (wt%) of total fatty acids in a stable phase 10 weeks after transplantation. Results: During a median follow-up time of 6,8 years, 569 (28,6%) renal allografts were lost, either due to patient death (n=340, 59.8% of graft loss) or graft loss in surviving patients (n=229, 40.2%). Plasma marine n-3 polyunsaturated fatty acid levels ranged from 1.35 to 23.87 wt%, with a median level of 7.95 wt% (interquartile range 6.20 to 10,03 wt%). When adjusting for established graft loss risk factors, there was a 1 1 % reduced risk of graft loss for every 1.0 wt% increase in marine n-3 polyunsaturated fatty acid level (adjusted hazard ratio 0.89; 95% confidence interval 0.84 to 0.93), and a 10% reduced risk of graft loss in surviving patients (adjusted hazard ratio 0.90; 95% confidence interval 0.84 to 0.97).

High levels of plasma marine n-3 polyunsaturated fatty acids were associated with better renal allograft survival.

Plasma marine n-3 PUFA levels ranged from 1.35 to 23,87 wt%, with an interquartile range of 6.20 to 10.03 wt% and a median level of 7.95 wt%. Patient characteristics for selected variables according to marine n-3 PUFA levels are given in Table 5 below. Patients with high marine n-3 PUFA levels were older, less often current smokers, had lower plasma n-6 PUFA levels and despite the higher age group, fewer had diabetes mellitus prior to transplantation. They were also more often treated with cyclosporin A than tacrolimus. Adult renal transplant recipients not included in the study were older (mean age 55.1 years) than study participants (mean age 51.6 years).

Table S: Baseline characteristics according to levels of marine n-3 polyunsaturated fatty acids

Table 5 provides the baseline characteristics of the study population according to marine n-3 polyunsaturated fatty acid (PUFA) levels, defined as the sum of plasma phospholipid eicosapentaenoic acid (EPA), docosapentaenoic acid and docosahexaenoic acid (DHA) levels in weight percentage (wt%) of total plasma phospholipid fatty acids. Results are presented as proportions for categorical data, median and interquartile range for time in dialysis and mean and standard deviations for other continuous data. Differences in baseline characteristics were evaluated using Mantel-Haenszel test of linear trend for categorical data, Kruskal-Wallis test for time in dialysis and linear regression for other continuous data. The presence of diabetes mellitus and coronary artery disease were recorded before renal transplantation. Recipient and donor age, time in dialysis and smoking status were recorded at the time of transplantation. Choice of calcineurin inhibitor were recorded, polyunsaturated fatty acid levels were determined and estimated glomerular filtration rate using the Modification of Diet in Renal Disease formula (eGFR) and albumin levels were measured at a clinical appointment 10 weeks post- transplant. During a median follow-up time of 6.8 years, 569 (28.6%) renal allografts were lost, either due to patient death (n=340, 59.8% of graft loss) or graft loss in surviving patients (n=229, 40.2%). In multivariable adjusted Cox proportional hazard regression analysis we found a significant negative association between marine n-3 PUFA levels and both overall and death censored graft loss (Table 6). For every 1 .0 wt% increase in marine n-3 PUFA level, there was a 1 1 % reduced risk of overall graft loss (adjusted hazard ratio [HR] 0.89; 95% confidence interval [CI] 0.84 to 0.93) and a 10% reduced risk of death censored graft loss (adjusted HR 0.90; 95% CI 0.84 to 0.97). Spline curve analysis showed that associations between marine n-3 PUFA levels and overall and death censored graft loss were linear with lower hazard for graft loss with higher marine n-3 PUFA levels, when adjusted for recipient age and n-6 PUFA levels (Figure 3). In subgroup analysis, the content of EPA, but not DHA, was associated with death censored graft loss (EPA levels: adjusted HR 0.81 ; 95% CI 0.71 to 0.93. DHA levels: adjusted HR 0.91 ; 95% CI 0.80 to 1 .02), whereas both were associated with overall graft loss (EPA levels: adj usted HR 0.84; 95% CI 0.77 to 0.91. DHA levels: adj usted HR 0.87; 95% CI 0.80 to 0.94). There was no association between DPA levels and neither overall nor death censored graft loss (data not shown). Acute rejection rates during the first tfiree months after renal transplantation did not differ between patients with high or low plasma levels of marine n-3 PUFAs. Beyond the first three months, patients belonging to the lowest marine n-3 PUFA quartile had significantly more acute rejection episodes ( i 1.7% in the lowest quartile versus 7.8% in the upper quartile). Similar results were found for EPA and DHA levels.

Table 6: Estimated risk of graft loss

Table 6 provides the estimated risk of overall and death censored graft loss, using multivariate adjusted Cox proportional hazard regression analysis. Shown are hazard ratios (HR) for developing graft loss endpoints for all the variables included in the Cox model after multivariable adjustment, including marine n-3 polyunsaturated fatty acid (PUFA) levels (the sum of eicosapentaenoic acid, docosahexaenoie acid and docosapentaenoic acid levels per 1 .0 weight percentage (wt%) increase (wt% total plasma phospholipid fatty acids), n-6 PUFA levels per 1.0 vvt% increase and pre-defined graft loss risk factors presented in the text and also shown in Table 6. For categorical variables the adjusted hazard ratio (HR) refers to the presence of a condition or treatment or the default alternative shown in brackets. For continuous variables the adjusted HR refers to the unit increase shown in brackets (e.g. per 0.1 g/dL increase in albumin level). Marine n-3 PUFA and n-6 PUFA levels were determined by gas chromatography at 10 weeks post-transplant. Recipient age, donor age, gender, number of human leukocyte antigen (HLA) DR mismatches, living or deceased donor, first or previous renal transplantation, smoking status, history of coronary artery disease, stroke, peripheral vascular disease or diabetes mellitus and transplant era ( 1999-2006 versus 2007-201 1 ) were registered at the time of transplantation. Estimated glomerular Filtration rate (eGFR) according to the

Modification of Diet in Renal Disease formula, total cholesterol and albumin were measured and the number of antihypertensive drugs, use of calcineurin inhibitors cyclosporin A or tacrolimus, use of induction therapy with the anti-interleukin-2 receptor antibody basiliximab, use of mammalian target of rapamycin (mTOR) inhibitors and any cytomegalovirus infections or acute rejection episodes within the early phase after transplantation were recorded at 10 weeks post-transplant. CI : confidence interval.

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Claims

Claims:

1. A method of administering a renal protective composition comprising n-3 PUFAs to a patient who has received, or who is scheduled to receive, a renal transplant.

2. A method as claimed in claim I wherein the method is for improving at least one parameter associated with renal function.

3. A method as claimed in any of the claims I or 2 to correct a deficiency or to increase the level of said n-3 PUFAs in the plasma phospholipid in the blood.

4. A method as claimed in any of the claims l to 3 wherein the level of n-3 PUFAs is increased to above about 8.0 wt% of the total plasma phospholipid fatty acid level in the blood.

5. A method as claimed in any of the claims 1 to 4 wherein the level of EPA is increased to > 1 ,5 wt% of the total plasma phospholipid fatty acid level in the blood,

6. A method as claimed in any of the claims 1 to 5 wherein the method corrects an imbalance in the ratio of n-6 to n-3 PUFAs in the blood.

7. A method as claimed in claim 6 wherein the ratio of n-6 to n-3 PUFAs in the blood is corrected to a ratio of less than about 4.0.

8. A method as claimed in claim 6 wherein the ratio of arachidonic acid to n-3 PUFAs in the plasma phospholipids is corrected to less than about 1.0.

9. A method as claimed in any of the claims 1 to 8 wherein the Omega-3 Index (%) is increased to above about 1 1.0, more preferably above about 13.0.

10. A method as claimed in any of the claims 1 to 9 wherein the composition is selected from the group of an active pharmaceutical ingredient (API), a food supplement, a dietary supplement, a nutritional supplement, over-the-counter (OCT) supplement, pharmaceutical grade supplement,

Enteral Formulas for Special Medical use. Foods for Specified Health Uses, Food for Special

Medical Purposes (FSMP), Food for Special Dietary Use (FSDU), Medical. Nutrition and a Medical food.

1 1 . A method as claimed in any of the claims 1 to 10 wherein the composition comprises at least 55 wt% of at least one of EPA and DHA by the weight of the fatty acids therein.

12. A method as claimed in any of the claims 1 to 1 1 wherein the composition comprises at least 75 wt% of at least one of EPA and DHA by the weight of the fatty acids therein.

13. A method as claimed in any of the claims 1 to 12 wherein the weight ratio of EPA: DHA in the composition ranges from about 1 : 10 to about 10; 1 , and more preferably 1 : 1 to about 2: 1 and most preferably from about 1.2 to about 1.3.

14. A method as claimed in any of the claims 1 to 13 wherein the risk of overall mortality and cause- specific mortality in RTRs, cardiovascular morbidity and mortality, myocardial infarction (Ml), sudden cardiac death (SCO), stroke or infectious disease is reduced or the renal function is improved.

15. A method as claimed in any of the claims 1 to 14 improving at least one parameter associated with renal function providing beneficial effects on either inflammation, renal fibrosis, endothelial function, lipid profile or blood pressure.

16. A method as claimed in any of the claims 1 to 15 wherein the renal function is improved,

stabilized, maintained or the decline is slowed down, identified as an improved or maintained GFR,

17. A method as claimed in any of the claims 1 to 16 reducing the risk of graft loss.

18. A composition comprising n-3 PUFAs as a renoprotective composition for renal transplant

recipients.

19. A composition as claimed in claim 18 for improving at least one parameter associated with renal function,

20. A composition as claimed in any of the claims 18 and 19 for use in the treatment or for dietary management of a patient who has received, or who is scheduled to receive, a renal transplant, to correct a deficiency or to increase the level said n-3 PUFAs in the blood.

21. A composition for use according to claim 20 wherein the level of n-3 PUFAs is increased to above about 8.0 wt% of the total plasma phospholipid fatty acid level in the blood.

22. A composition for use according to claims 20 or 21, wherein the level of EPA is increased to > 1.5 wt% of the total plasma phospholipid fatty acid level in the blood.

23. A composition for use according to any of claims 20 to 22 wherein the Omega- 3 Index (%) is increased to above about 1 1.0, more preferably above about 13.0.

24. A composition for use according to any of the claims 20 to 23 wherein the ratio of n-6 to n-3 PUFAs in the blood is corrected to a ratio of less than about 4.0.

25. A composition for use according to any of the claims 20 to 23 wherein the ratio of arachidonic acid to n-3 PUFAs in the plasma phospholipids is corrected to less than about 1.0.

26. A composition according to any of the claims 18 to 23 comprising at least 55 wt% of at least one of EPA and DHA by the weight of the fatty acids therein .

27. A composition according to any of the claims 18 to 26 wherein the composition is selected from the group of a pharmaceutical composition, a food supplement, a dietary supplement, a nutritional supplement, over-the-counter (OCT) supplement, pharmaceutical grade supplement, Enteral Formulas for Special Medical use. Foods for Specified Health Uses, Food for Special Medical

Purposes (FSMP), Food for Special Dietary Use (FSDU), Medical Nutrition and a Medical food.

28. A composition according to any of the claims 18 to 27 which provides a reduced risk of overall mortality and cause-specific mortality in RTRs, cardiovascular morbidity and mortality, myocardial infarction (MI), sudden cardiac death (SCD), stroke or infectious disease, or which improves renal function.

29. A composition according to any of the claims 18 to 28 which reduces the risk of graft loss,

30. A composition according to aoy of the claims! 8 to 28 improving at least one parameter associated with renal function providing beneficial effects on either inflammation, renal fibrosis, endothelial function, lipid profile or blood pressure.

31.. A composition according to any of the claims 18 to 28 wherein the renal function is improved, maintained or the decline is slowed down, identified as an improved or maintained GFR.