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Volume: 12 Issue: 6 December 2014


Supplementation With Omega-3 Polyunsaturated Fatty Acids and Experimental Tacrolimus-Induced Nephrotoxicity

Objectives: Calcineurin inhibitors are effective immunosuppressive agents, but associated adverse effects such as nephrotoxicity may limit efficacy. Dietary fish oil may minimize nephrotoxicity caused by long-term use of calcineurin inhibitors. The purpose of the present study was to evaluate the effects of omega-3 fatty acids on calcineurin inhibitor nephrotoxicity in rats that had normal kidney function or chronic kidney failure.

Materials and Methods: Rats that had normal kidney function or chronic renal failure that was induced by mass reduction surgery were treated with tacrolimus without or with fish oil, fish oil alone, or olive oil. Kidney function and histology were evaluated after 14 days.

Results: Mean body weight loss, serum creatinine, change in serum creatinine, and rate of decrease in creatinine clearance were greater in normal rats that received than did not receive tacrolimus. Tacrolimus nephrotoxicity was greater in rats that had chronic renal failure than normal kidney function, but the mean change in serum creatinine was significantly lower in rats with chronic renal failure that were treated with tacrolimus and fish oil than tacrolimus alone. Fish oil supplementation was associated with fewer abnormal histo-pathologic lesions in the kidneys of tacrolimus-treated rats that had normal kidney function or chronic renal failure (not signifant).

Conclusions: Fish oil may be protective against the development of kidney dysfunction and histo-pathologic changes in rats treated with tacrolimus.

Key words : Adverse events, Calcineurin inhibitors, Chronic renal failure, Immunosuppression, Fish oil


Calcineurin inhibitors are effective immuno-suppressive drugs for kidney transplant, but efficacy of these drugs may be decreased because of nephrotoxicity.1 Calcineurin inhibitor toxicity has increased the prevalence of chronic kidney disease in recipients of other types of solid-organ transplants.2 Posttransplant kidney dysfunction is related to meaning after transplant kidney function and the amount of functional at transplant.3 Development of chronic kidney dysfunction after kidney and other solid-organ transplant may increase morbidity and mortality.4 Protocols that do not include calcineurin inhibitors may be associated with adverse events and limited graft survival. Alternative immuno-suppressive protocols are being evaluated with nonnephrotoxic drugs in which calcineurin inhibitors are avoided, discontinued, or minimized.5,6

Omega-3 fatty acids may benefit transplant recipients by decreasing the development of rejection episodes and hypertension, but studies of the effects of omega-3 fatty acids on calcineurin inhibitor toxicity are limited.7-10 Fish oils contain large amounts of polyunsaturated omega-3 fatty acids, may suppress proinflammatory activity, and may promote vasodilation.8

The purpose of the present study was to evaluate the effects of omega-3 fatty acids on calcineurin inhibitor nephrotoxicity in rats that had normal kidney function or chronic kidney failure because of reduced kidney mass. We hypothesized that dietary supplementation with omega-3 fatty acids may minimize the development of minimize calcineurin inhibitor nephrotoxicity. We also hypothesized that in rats receiving nonrenal solid-organ transplants, renal remnant mass may be inversely related to the frequency of calcineurin inhibitor toxicity, and rats that have less renal remnant mass may have may improved nephrotoxicity from omega-3 fatty acid supplementation.

Materials and Methods

Adult male Wistar rats (250-350 g) were housed individually in metabolic cages to permit collection of urine. The cages were maintained in a temperature- and light-controlled environment and the rats were allowed free access to water. The animal procedures were performed in accordance with protocols that were approved by the Institutional Animal Care and Use Committee of the Medical School Sao Jose do Rio Preto (FAMERP).

Tacrolimus (Prograf, Janssen Cilag, Sao Paulo, Brazil) was injected subcutaneously into the rats once daily (3 mg/kg/d).11 Fish oil (0.5 mL) that contained eicosapentaenoic acid (90 mg) and docosahexaenoic acid (60 mg) was administered once daily by oral gavage (ProEPA [1000 mg/mL], Aché Laboratórios Farmacêuticos S.A., Guarulhos, SP, Brazil) during the 14-day study.

Experimental design
Rats (n = 56) were randomly divided into 2 groups (28 rats each): (1) normal rats that had 2 kidneys and 100% renal mass (2K), and (2) rats that had chronic renal failure that was induced by a 5/6 renal mass reduction (5/6K) that was performed as previously described.12 The 2 groups each were subdivided into 4 treatment groups (7 rats each): (1) tacrolimus alone; (2) fish oil alone; (3) combination tacrolimus and fish oil; and (4) olive oil. At 0 and 14 days after surgery, we measured urinary volume, body weight, serum creatinine, creatinine clearance, and 24-h proteinuria. The change in serum creatinine was calculated as the difference between serum creatinine at 0 and 14 d, and the rate of decrease in clearance creatinine was calculated. At 14 days, the animals were killed and the kidneys were evaluated with histology.

Kidney function and tacrolimus blood level
Plasma and urine creatinine concentrations were determined using a colorimetric assay (Jaffe method) (Spectrophotometer BTS 310, Biosystems S.A., Barcelona, Spain). Tacrolimus blood levels were determined by double antibody radioimmunoassay.13 Creatinine clearance was calculated from mea-surements of serum and urinary creatinine levels.

All histologic analyses were performed by an observer who was blinded to treatment. Coronal sections of the kidney were fixed by immersion in paraformaldehyde solution and embedded in paraffin. Light microscopy was performed on 3-mm sections of tissue stained with hematoxylin-eosin and periodic acid methenamine silver to assess glomerulosclerosis and tubulointerstitial fibrosis. Glomerulosclerosis was defined as glomeruli with sclerosis or mesangial expansion with tuft adherence. A minimum 50 glomeruli per rat kidney were evaluated, and the mean value was used as representative for the rats. Glomerular sections were assessed by semiquantitative analysis.14 The extent of glomerulosclerosis was graded from 0 to 4 (0, normal glomeruli; (1), mesangial thickening < 25% of the tuft; (2), moderate glomerulosclerosis with mesangial proliferation and thickening ≤ 50%; (3wW7, severe glomerulosclerosis with obliteration of capillaries and diffuse sclerosis ≤ 75%; and (4), complete capillary obliteration and thrombosis with global sclerosis ≤ 100%).

Tubulointerstitial fibrosis damage was evaluated by semiquantitative analysis of 20 cortical fields. Tubulointerstitial damage was graded according to the extent (%) of tubular damage (infiltration, fibrosis, and tubular dilatation or atrophy) within the evaluated fields from the renal cortex.15

Statistical analyses
Data analysis was performed with statistical software (Prism, GraphPad Software Inc., San Diego, CA, USA). Data were reported as mean ± standard deviation (SD). Comparisons of data between groups were performed with analysis of variance and Tukey test. Changes in body weight within each group during the study were evaluated with paired t test. Statistical significance was defined by P < .05.


Tacrolimus blood levels
Mean tacrolimus blood levels were similar between the groups that received tacrolimus (2K [tacrolimus], 16 ± 5 ng/mL; 5/6K [tacrolimus], 11 ± 4 ng/mL; 2K [tacrolimus and fish oil], 12 ± 3 ng/mL; and 5/6K [tacrolimus and fish oil], 14 ± 5 ng/mL; not significant).

Effect of tacrolimus and fish oil on kidney function
Mean body weight loss, serum creatinine, change in serum creatinine, and rate of decrease in creatinine clearance were greater in normal rats (2K) that received than did not receive tacrolimus (Table 1). Body weight loss was significantly less in the 2K rats that received tacrolimus and fish oil than tacrolimus alone (Table 1).

The effect of nephrotoxicity of tacrolimus in rats that had chronic renal failure (5/6K) was greater than normal rats (2K). In 5/6K rats, all treatment subgroups had decreased body weight after treatment (Table 1). The mean change in serum creatinine was significantly lower in 5/6K rats treated with tacrolimus and fish oil than tacrolimus alone (Table 1). No significant differences in mean serum creatinine, rate of decrease in creatinine clearance, or 24-hour proteinuria were observed between treatment subgroups in 5/6K rats.

Effect of fish oil and tacrolimus nephrotoxicity on the remnant kidney mass
To evaluate the effects of fish oil supplementation on tacrolimus effects in the remnant kidney mass, we compared the change in serum creatinine and rate of decrease in creatinine clearance between rats treated with either tacrolimus alone or tacrolimus and fish oil (Figure 1). The mean change in serum creatinine was 3-fold greater in the 5/6K than 2K groups (Figure 1). The mean rate of decrease in creatinine clearance was similar in 5/6K and 2K rats that were treated with tacrolimus only (2K, 0.04 ± 0.02; 5/6K, 0.037 ± 0.008; not significant), but was 3-fold greater in 5/6K than 2K rats that were treated with tacrolimus and fish oil (2K, 0.02 ± 0.01 mL/min/d; 5/6K, 0.052 ± 0.006 mL/min/d; P ≤ .02) (Figure 1).

Tacrolimus nephrotoxicity affected 5/6K and 2K rats, but greater scores in tubulointerstitial damage and glomerular damage index were observed in the kidneys of 5/6K than 2K rats (Figure 2). Fish oil supplementation was associated with fewer tubulo-interstitial and glomerular lesions in both 5/6K and 2K rats that were treated with tacrolimus (Figure 3).


Tacrolimus is the most commonly used calcineurin inhibitor in patients who have solid-organ transplant, but the use of tacrolimus is associated with progressive nephrotoxicity. Therefore, immuno-suppressive programs to minimize or withdraw calcineurin inhibitors have been proposed for kidney transplant.16 In recipients of solid-organ transplants other than kidney transplants, the nephrotoxic effect of calcineurin inhibitors may cause chronic kidney disease and increase morbidity and mortality.17-19 The present results showed that serum creatinine level alone did not adequately quantify the changes in kidney function that were observed, similar to findings of previous studies.20,21 The change in serum creatinine and rate of decrease in creatinine clearance were more effective than serum creatinine at showing beneficial effects of dietary fish oil supplementation on kidney function.

Independent of kidney mass, all groups had increased mean serum creatinine and weight loss after treatment for 2 weeks with tacrolimus, especially rats that had chronic renal failure. The nephrotoxic effects of tacrolimus were greater in rats that had chronic renal failure (5/6K); these rats had greater mean weight loss, higher mean serum creatinine level, lower mean creatinine clearance, and greater mean 24-hour proteinuria than 2K rats. The differences observed between the 5/6K and 2K rats may have been caused by surgical and hemodynamic effects that occur after the reduction of kidney mass and may have been exacerbated by tacrolimus nephrotoxicity.

Fish oil supplementation in 2K rats was associated with significantly lower weight loss, 2-fold lower variation in serum creatinine, and approximately 50% lower rate of decrease of creatinine clearance. The effect of fish oil supplementation on the histopathology was evident in the analysis of tubulointerstitial disease, despite the short duration of the study. The renoprotective effects of long-chain omega-3 fatty acids derived from fish oil (eicosapentaenoic acid and docosahexaenoic acid) may be attributed to anti-inflammatory properties that may include decreased production of inflammatory mediators, a block of signaling cascades that are important in the generation and expression of proinflammatory cytokines, and a block of angiotensin II signaling in the kidney.10,22,23

Remaining nephron mass may act synergistically with polyunsaturated fatty acids in vivo. Therefore, recipients of nonrenal solid-organ transplants who have pretransplant kidney dysfunction may benefit from fish oil supplementation that may attenuate calcineurin inhibitor nephrotoxicity. Furthermore, in recipients of kidney transplants who have tacrolimus immunosuppression, fish oil supplementation may help minimize the progression of chronic allograft dysfunction.

In summary, we showed that orally administered fish oil containing omega-3 fatty acids may be associated with a decrease in tacrolimus nephro-toxicity in rats that have normal or abnormal kidney function. Further study may evaluate whether transplant patients may benefit from the renoprotective effects of omega-3 fatty acids on the remaining kidney mass.


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Volume : 12
Issue : 6
Pages : 522 - 527
DOI : 10.6002/ect.2014.0116

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From the 1Laboratory of Immunology and Experimental Transplantation-LITEX; the 2Department of Medicine, Division of Nephrology, Medical School of Sao Jose do Rio Preto Foundation of Hospital de Base (FAMERP/FUNFARME); and the 3Instituto de Urologia e Nefrologia, Sao Jose Rio Preto, SP, Brazil
Acknowledgements: The authors declare that they have no sources of funding for this study, and they have no conflicts of interest to declare.
Corresponding author: Mario Abbud-Filho, PhD, MD, Department of Medicine, Laboratory of Immunology and Transplantation Experimental – LITEX, Av. Brigadeiro Faria Lima 5416- 15090-000, São Jose do Rio Preto, SP, Brazil
Phone: +55 17 3201 5739
Fax: +55 17 4009 9157