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Effects of Immunosuppressive Drugs on Serum Fatty Acids of Phospholipids Fraction in Renal Transplant Recipients
Effects of Immunosuppressive Drugs on Serum Fatty Acids of Phospholipids Fraction in Renal Transplant Recipients M. Wilusza,*, D. Cieniawskib, J. Bugajskaa, J. Berskaa, E. Ignacakb, A. Bętkowska-Prokopb, M. Kuzniewskib, W. Sułowiczb, and K. Sztefkoa a
Department of Clinical Biochemistry, and bDepartment of Nephrology, Jagiellonian University Medical College, Cracow, Poland
ABSTRACT Background. Immunosuppressive medications often cause posttransplant hyperlipidemia. The effects of cyclosporine (CsA) and tacrolimus (Tac) on lipid profile is wellknown; however, there are very few studies related to the effect of these immunosuppressants on fatty acids (FA) of phosholipids fraction (PL) in renal transplant recipients (RTR). We sought to analyze the FA profile in PL fraction of RTR treated with Tac or CsA. Methods. The study included 65 renal transplant patients on CsA (n ¼ 24, group I) or Tac (n ¼ 41, group II), and 14 healthy controls. Individual serum FA concentrations were measured by gas chromatography. Chemstation software was used to analyze the data. Results. No differences between studied groups and controls were noted for monounsaturated FA, polyunsaturated n-3 FA (PUFA n-3), PUFA n-6, or the ratio of PUFA n-6 to PUFA n-3. The following mean values of FA were significantly higher in the CsA-RTR and Tac-RTR as compared with controls: total FA (P < .01 in both cases), saturated FA (SFA; P < .02 in both cases), C12 (P < .003 in both cases), C18 (P < .003 in both cases), and C18:2 (P < .01 for CsA RTR; P < .02 for Tac RTR). No differences between the measurements in patients on CsA and in patients on Tac were noticed. Significant correlation between SFA and eGFR was observed only in the CsA RTR group (P < .05). A negative relationship between PUFA n-6 and the estimated glomerular filtration rate was seen, but the correlation was not significant. Conclusions. Immunosuppressive drugs may affect FA metabolism, but the FA profile does not depend on the type of immunosuppressive drug administered.
P
ROGRESS in immunosuppressive management of patients after organ transplantation has significantly increased their overall survival. Tacrolimus (Tac) and cyclosporine (CsA) are among the most commonly used immunosuppressive drugs. These medications may, however, cause different side effects, among which lipid metabolic disorders are most frequently observed [1e4]. It has been shown that in patients receiving Tac the triglyceride (TG) levels are lower as compared with patients prescribed with CsA [3,5]. In contrast, treatment of the organ transplant patients with CsA causes significant increase of total cholesterol and low-density lipoprotein cholesterol levels [2,3,6]. Disturbances in lipid metabolism may also contribute to glomerular and interstitial injury that in turn
can promote renal disease progression. Posttransplant hyperlipidemia has been linked to cardiovascular disease, leading to a high morbidity and mortality rate [7,8]. Hyperlipidemia not only leads to development of atherosclerosis of coronary arteries, but also of renal artery, which can affect graft function [9,10]. Accumulation of lipids in nonadipose tissues may lead to cellular dysfunction and cell death, the phenomenon called lipotoxicity [11]. It has been confirmed that saturated fatty *Address correspondence to Małgorzata Wilusz, Department of Clinical Biochemistry, Jagiellonian University Medical College, University Children’s Hospital of Cracow, Wielicka 265, 30-663 Cracow, Poland. E-mail: [email protected]
0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.03.026
ª 2016 Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
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Transplantation Proceedings, 48, 1616e1622 (2016)
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acids (SFA), mainly palmitic acid, can be responsible for lipotoxicity of most cells, including renal cells (podocytes) [12]. Unlike SFA, it has been shown that monounsaturated fatty acids (MUFA) have protective effect on renal lipotoxicity, but the mechanism has not been fully understood yet. It is believed that the presence of oleic acid in podocytes can facilitate incorporation of palmitic acid into TG. Because of that lower level of intracellular concentration of palmitic acid is seen. On the other hand, increased rate of the b-oxidation of palmitic acid prevent accumulation of SFA in the cell [13,14]. Another class of fatty acids (FAs)dthe n-3 FAdmay protect kidney function by modulating the inflammatory response through downregulation of proinflammatory cytokines production, cyclooxygenase-2 activity, and an expression of endothelial leukocyte adhesion molecules [15e17]. In contrast, progressive deterioration of kidney function after dietary supplementation with n-6 FA has been shown [17]. Eicosanoids, which are the products of arachidonic acids (AAs) metabolism, have a wide range of biological action in inflammatory processes and immunity [18,19]. According to Brown et al. [20], the production of vasoactive eicosanoids may contribute to glomerular hyperfunction and consequently to glomerular damage. Dyslipidemia may also affect kidney function indirectly through vascular injury and oxidative stress. Cristol at al. [21] suggested that oxidation of polyunsaturated FAs (PUFA) may play a significant role in the vascular lesions. There are few studies related to the effect of different immunosuppressants on FA patterns in renal transplant recipients. Such findings would be significant, because FA play an important role in renal homeostasis and renal function. Furthermore, it may be speculated that changes in dietary habits towards higher intake of unsaturated FA could prevent or delay the progression of dyslipidemiarelated renal disease.
MATERIALS AND METHODS The study included 65 renal transplant recipients on immunosuppressive therapy: 24 patients treated with CsA (9 females, 15 males; mean age, 54 12 years [range, 25e65]) and 41 patients treated with Tac (15 females, 26 males; mean age, 51 13 years [range, 27e70]). All patients underwent renal transplantation, and were treated at the Department of Nephrology, Jagiellonian University Medical College, Cracow, Poland. The average time since transplantation was 42 months (range, 15e120). Therapeutic regimens were established individually for each patient, taking into account immunologic risk, comorbidity, ischemia time, and clinical experience. Safety and efficacy assessments were performed at scheduled study visits and therapy adjustments were introduced if necessary. Only patients who maintained their originally assigned calcineurin inhibitor were included in the study. The majority of patients received triple drug immunosuppressive therapy with a calcineurin inhibitor (CsA or Tac), mycophenolic acid, and glucocorticoids. Renal transplant recipients were treated with prednisone (in CsA group: 20 patients; in Tac group: 29 patients), methylprednisolone (in CsA group: 3 patients; in Tac group: 6 patients), and prednisolone (2 patients in Tac group). Five patients did not receive steroid therapy during the survey (1 in CsA group and 4 in Tac group). Each patient was 1 year posttransplant. Regardless of the calcineurin inhibitor used, all patients received low doses of steroids (average dose of glucocorticoids based on prednisone in CsA group, 6.66 mg; in Tac group, 5.19 mg). Furthermore, in patients of both groups glucocorticoids were used at comparable doses; thus, the effect of steroids was not taken into account in the statistical analysis. Patients with diabetes mellitus before renal transplantation, and the patients with a glomerular filtration rate (GFR) of <30 mL/min were excluded. The control group consisted of 14 healthy volunteers (3 males and 11 females; mean age, 48 12 years [range, 26e69]). The study protocol was approved by the Bioethics Committee of the Jagiellonian University and written informed consent was obtained from all patients. Baseline demographics and clinical characteristics of patients are presented in Table 1. Fasting blood samples for serum FA of PL determination were obtained from each patient. The blood serum was separated and
Table 1. Baseline Demographics and Clinical Characteristics of Patients Characteristic
Cyclosporine Group (n ¼ 24)
Tacrolimus Group (n ¼ 41)
Age range (y) Mean age (y) Male Female eGFR, mean (range), mL/min BMI, mean (range), kg/m2 Acute graft rejection Diabetes type II (after transplantation) Hypertension Lipid profile, mean (range), mmol/L TC TG LDL-C HDL-C Drug levels at the time of measurement, mean (range), ng/mL
25e65 54 12 15 9 65.3 (35.3e119.9) 26.7 (17.8e33.2) 2 8 8
27e70 51 13 26 15 75.4 (31.8e126.5) 26.8 (19.7e38.1) 3 8 15
4.97 (3.4e7.0) j 41.7%* 1.93 (0.53e3.3) j 33.3%* 2.95 (1.2e5.0) j 20.8%* 1.45 (0.7e2.1) j 12.5%* 452.00 (60.7e1167.5)
4.80 (3.0e6.9) j 43.9%* 1.89 (0.6e3.6) j 39.0%* 2.66 (1.5e4.1) j 21.9%* 1.32 (0.9e2.5) j 12.2%* 6.05 (3.9e11.5)
Abbreviations: BMI, body mass index; eGFR, estimated glomerular filtration rate; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides. *Percent of results above (TC 5 mmol/L; TG 1.7 mmol/L; LDL 3 mmol/L) or below (HDL <1 mmol/L for men, <1.2 mmol/L for women) recommended values.
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Table 2. Mean Values of Serum Fatty Acids of Phospholipids Fraction Concentration (mmol/L) in Renal Transplant Recipients Treated With Cyclosporine or Tacrolimus FA
SFA C12 C14 C16 C18 C24 MUFA C16:1 C18:1 PUFA C18:2 C20þC18:3(n-6) C18:3 C20:2 C20:4 C20:5 C22:6 PUFA n-3 PUFA n-6 PUFA n6/PUFA n3 SFA MUFA Total FA
Cyclosporine Group
3.92 17.51 1130.53 441.06 26.14
0.42† 0.94 31.17 18.61† 1.33
14.22 1.09 289.97 12.78 593.50 16.72 8.00 18.47 546.28 40.74 304.05 348.54 1175.00 3.56 1599.35 302.48 3463.61
25.98* 0.72 0.61 1.00 28.09 4.27 19.03 21.20 38.15 0.20 44.75** 11.76 93.52*
Tacrolimus Group
5.17 19.87 1117.93 436.29 27.02
0.39† 0.95 30.14 12.42† 1.07
18.10 1.46 297.37 12.05 594.04 15.94 8.33 19.21 561.25 52.07 297.73 343.04 1157.04 3.53 1564.38 318.03 3378.70
15.70** 0.66 0.43 0.61 18.54 3.09 13.43 40.53 25.67 0.17 38.27** 13.48 88.15*
Control Group
1.86 17.77 1011.09 359.40 27.10
0.27 2.40 65.31 21.23 1.83
19.80 4.80 289.54 32.54 503.80 15.23 8.33 18.48 523.72 51.55 260.70 315.87 1074.17 3.97 1388.26 293.86 2953.54
29.61 1.16 1.49 1.72 37.91 7.65 29.70 40.53 45.65 0.53 82.07 33.83 130.05
Values are presented as the mean SE (95% confidence interval). Abbreviations: FA, fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids. *P < .01; **P < .02; †P < .003 compared with the control group.
kept frozen at 70 C until measurement. The following analytical steps were taken: lipids extraction from serum with the use of Folsch method [22], separation of lipid fractions, and methylation of FA of PL fraction. Separation of the FA methyl esters was performed using gas chromatography equipped with flame ionization detector (Agilent Technologies 6890 Network GC System). The following FA were measured: myristic acid (C14), palmitic acid (C16), stearic acid (C18), palmitoleic acid (C16:1cis [n-7]), oleic acid (C18:1cis [n-9]), linoleic acid (LA; C18-2cis [n-6]), a-linolenic acid (ALA; C18:3cis [n-3]), 11,14-eicosadienoic acid (20:2cis [n-6]), AA (C20:4cis [n-6]), eicosapentaenoic acid (EPA; C20:5cis [n-3]), docosahexaenoic acid (DHA; C22:6cis [n-3]), and lignoceric acid (C24). Chemstation software was used to analyze the data. Total FA, total SFAs, total MUFAs, the sum of PUFA n-6, and the sum of PUFA n-3 concentrations, as well as the ratio of PUFA n-6 to PUFA n-3, were calculated. All results were expressed as mmol/L.
Statistical Analysis Descriptive statistics including mean values and standard error were calculated. To compare the FA profiles between patients on CsA or Tac and control group, one-way analysis of variance with a post hoc Newman-Keul test was applied. Pearson’s correlation coefficients were used to establish the relationship between FAs (PUFA n-6, PUFA n-3, total FA, SFA, and MUFA) and the estimated GFR (eGFR; calculated according to Cockcroft-Gault formula). The Student t test was performed to compare the differences in FA profiles between male and female patients. All analyses were done with the use of Statistica 10 (StatSoft). Statistical significance was set for P < .05.
RESULTS
The mean values (standard error) of individual FA of PL, as well as of total FAs, and the sum of: MUFA, SFA, PUFA n-3 and PUFA n-6, in renal transplant recipients receiving CsA or Tac and control group, are shown in Table 2. No differences between all measurements in patients on CsA and patients on Tac were observed. Patients treated with CsA and patients treated with Tac had significantly higher the mean SFA levels (P < .01 for CsA; P < .02 for Tac) as compared with controls. In both patient groups, the mean values of C12 and C18 were significantly higher than the mean value observed in control (P < .003 in both cases). The mean values of palmitic acid level were slightly higher in patient groups than in control; however, the differences were not significant. No differences between patient groups and controls were noted for C16:1 and C18:1, or for the sum of MUFA. Among PUFA, only the mean values of C18:2 were significantly higher in the CsA and Tac groups, as compared with control (P < .009 for CsA; P < .02 for Tac). The mean levels of C18:3, C20:2, C20:4, and C20:5 in both patient groups were similar to the values obtained in the control group. No differences between the studied groups and the control were noted for mean values of C22:6, PUFA n-3, and PUFA n-6; however, the highest values were obtained for the patient groups. The mean values of the ratio of PUFA n-6 to PUFA n-3 in the CsA and Tac groups and in control were similar. Total FA level was statistically higher in the transplanted patients than in the control group
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Fig 1. Relationship between polyunsaturated fatty acids (PUFA) n-6 concentration and estimated glomerular filtration rate (eGFR) in the cyclosporine (CsA; A) and tacrolimus (Tac; B) groups. Relationship between saturated fatty acid (SFA) concentration and eGFR in the CsA (C) and Tac (D) groups. Relationship between monounsaturated fatty acid (MUFA) concentration and eGFR in the CsA (E) and Tac (F) groups.
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(P < .01 for both groups). No differences in FA profile were observed between the male and female in CsA and Tac groups. No correlation between total FA concentration and eGFR in either patient group was noted. In addition, no correlation between PUFA n-3 and eGFR in the patient groups was observed. There was a negative relationship between PUFA n-6 and eGFR in CsA and Tac groups, but the correlations were not significant (Fig 1A, B). In contrast, significant correlation between SFA and eGFR in CsA group (Fig 1C) was observed, but not in the Tac group (Fig 1D). MUFA was related negatively in the CsA group (Fig 1E) and positively to eGFR in the Tac group (Fig 1F). However, the relationship was not significant. DISCUSSION
The different FA have distinct effects on cell’s metabolism and its function. It should be stressed that mainly SFA and their metabolites are responsible for induction of lipotoxicity [13,23]. In the present study, a higher level of SFA of PL in patients after renal transplantation was observed as compared with the control group thus confirming its possible role in podocytes lipotoxicity. In contrast, MUFA have protective effect on renal injury. Oleic acid can prevent SFA-induced lipotoxicity by facilitating incorporation of palmitic acid into TG. Furthermore, oleic acid stimulates palmitic acid b-oxidation leading to reducing the level of palmitic acid [13,14]. In our study, MUFA concentration in patients on immunosuppressive therapy matched the results in the control group. Similar findings were presented in the paper on MUFA content in low-density lipoprotein fraction in patients after renal transplantation [24] and in the research on rats with [25] and without [26] immunosuppressive therapy. In contrast, the level of oleic acid in erythrocyte membrane in kidney transplant recipients was lower, although palmitoleic acid and MUFA were significantly higher than in the control [27]. According to Alexander et al [28], supplementation with oleic acid was highly effective in longer allograft survival time in animals treated with CsA. There is little doubt that MUFA have protective role in renal transplant recipients. It is believed that immunosuppressive drugs such as CsA and Tac can influence the biosynthesis of different PUFA through modulation of desaturation and elongation processes, by decreasing D9 desaturase, and increasing D5 and D6 desaturase activity [29]. In contrast with our previous study performed in patients after heart transplantation [30], in the present study no differences in PUFA n-3 levels between patient and control groups were observed. The results are not in agreement with those of others [24,25,31]. It was demonstrated that PUFA n-3 reduces inflammation [32e34]; however, in some papers the association between PUFA n-3 and inflammation has not been confirmed [35]. It is known that PUFA n-6 have proinflommatory properties. AA, a substrate of eicosanoids synthesis, initiates the
WILUSZ, CIENIAWSKI, BUGAJSKA ET AL
inflammatory process. It is believed that CsA nephrotoxicity is associated with specific alterations in renal AA metabolism [36]. The role of AA metabolites in kidney dysfunction and acute rejection has been reported [19,25]. In the present study, the level of AA as well as the total PUFA n-6 were similar in patients and in the control groups. This is in agreement with the data obtained by Baggio et al. [31], Cofan et al. [24], and Oh et al. [27]. In contrast, a lower content of AA was noticed in children after renal transplantation [37] and in transplanted rats treated with different immunosuppressants [25]. In patients after renal transplantation, the depletion of LA in erythrocyte membrane and in low-density lipoprotein fraction was reported, although the differences were not significant. In the present study, significantly higher levels of LA in patient groups were observed. Based on recent findings, LA could have both proinflammatory [38] and antiinflammatory properties. Higher levels of LA correlates with decrease of IL-6 level [35,39] and is linked to a lower C-reactive protein concentration [40]. It was proved that an increase in circulating LA is associated with reduced inflammation and diminished cardiovascular risk [41]. The reported higher LA level in the present study seems to be beneficial for transplant recipients. The balance between PUFA n-6 and PUFA n-3 in the diet has changed substantially over the years. Currently, consumption of n-6 FA predominates. Because PUFA n-6 and n-3 compete for the same enzymes involved in FA desaturation and elongation, a higher n-6/n-3 ratio correlates positively with the level of proinflammatory cytokines [39]. Liu et al. [42] reported that increased PUFA n-3/n-6 ratio were associated with decreased serum TG and total cholesterol levels, improved insulin resistance and reduced proinflammatory cytokine production. The similar n-6/n-3 ratio in patient and in control groups observed in our study does not contradict the previous research because transplant patients are more cautious about their diet. The eGFR is accepted as the best overall measure of kidney function, and facilitates the detection, evaluation, and management of chronic kidney disease. An increased risk of complications is related directly to lower eGFR values. Recent epidemiological studies have demonstrated that serum PUFAs levels correlate with lower rates of creatinine clearance in an older population [32]. Our study showed that patients with low eGFR treated with CsA or Tac had higher PUFA n-6 concentrations, although the correlation was not significant. It could support the previous data on the effects of dietary PUFA n-6 supplementation on glomerular hemodynamic and renal injury, which showed that dietary supplementation with PUFA n-6 may hasten renal injury [20,43,44]. Additionally, it was observed that increased eGFR was accompanied by different MUFA levels, depending on the immunosuppressant used. In patients treated with CsA, the level of MUFA decreased, whereas in patients treated with Tac, MUFA concentration increased. The role of MUFA in the nephroprotection is documented [11,13,14]. However, the reason for different
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effect of Tac and CsA on MUFA level is unknown. Similarly, the reason for difference in SFA level in relation to immunosuppressive therapy is unclear. A significant, negative correlation between eGFR and the level of SFA was only observed in patients treated with CsA. The toxicity of SFA on kidney function has already been described in literature [11,13,45]. In conclusion, it was confirmed that immunosuppressive drugs may affect FA metabolism in kidney transplant patients but the FA pattern did not depend on the type of immunosuppressive drug administered. The associations between the levels of PUFA n-6, MUFA, SFA, and eGFR indicate that FA play an important role in graft function in renal transplant patients. REFERENCES [1] Carta P, Zanazzi M, Di Maria L, et al. 5 year comparison of very low-dose cyclosporine and high-dose everolimus vs standard cyclosporine and enteric-coated mycophenolate in renal transplantation patients. Transplant Proc 2014;46:2228e30. [2] Ichimaru N, Takahara S, Kokado Y, et al. Changes in lipid metabolism and effect of simvastatin in renal transplant recipients induced by cyclosporine or tacrolimus. Atherosclerosis 2001;158: 417e23. [3] Colak T, Karakayali H, Yagmurdur MC, et al. Effect of conversion from cyclosporine to tacrolimus on lipid profiles in renal transplant recipients. Transplant Proc 2002;34:2081e2. [4] Mucha K, Foroncewicz B, Paczek L, et al. 36-month followup of 75 renal allograft recipients treated with steroids, tacrolimus, and azathioprine or mycophenolate mofetil. Transplant Proc 2003;35:2176e8. [5] Kanbay M, Yildirir A, Akcay A, et al. Effects of immunosuppressive drugs on serum lipid levels in renal transplant recipients. Transplant Proc 2006;38:502e5. [6] Akman B, Uyar M, Afsar B, et al. Lipid profile during azathioprine or mycophenolate mofetil combinations with cyclosporine and steroids. Transplant Proc 2007;39:135e7. [7] Sgambat K, He J, McCarter RJ, et al. Lipoprotein profile changes in children after renal transplantation in the modern immunosuppression era. Pediatr Transplant 2008;12:796e803. [8] Stoumpos S, Jardine AG, Mark PB. Cardiovascular morbidity and mortality after kidney transplantation. Transpl Int 2015;28: 10e21. [9] Massy ZA. Hyperlipidemia and cardiovascular disease after organ transplantation. Transplantation 2001;72(6 Suppl):S13e5. [10] Kimak E, Hałabis M, Baranowicz-Gaszczyk I. Relationships between serum lipid, lipoprotein, triglyceride-rich lipoprotein, and high-density lipoprotein particle concentrations in post-renal transplant patients. J Zhejiang Univ Sci B 2010;11:249e57. [11] Bobulescu IA. Renal lipid metabolism and lipotoxicity. Curr Opin Nephrol Hypertens 2010;19:393e402. [12] Kampe K, Sieber J, Orellana JM, et al. Susceptibility of podocytes to palmitic acid is regulated by fatty acid oxidation and inversely depends on acetyl-CoA carboxylases 1 and 2. Am J Physiol Renal Physiol 2014;306:F401e9. [13] Sieber J, Jehle AW. Free Fatty acids and their metabolism affect function and survival of podocytes. Front Endocrinol (Lausanne) 2014;5:186e7. [14] Listenberger LL, Han X, Lewis SE, et al. Triglyceride accumulation protects against fatty acid-induced lipotoxicity. Proc Natl Acad Sci U S A 2003;100:3077e82. [15] Donadio JV. n-3 Fatty acids and their role in nephrologic practice. Curr Opin Nephrol Hypertens 2001;10:639e42. [16] Tatsioni A, Chung M, Sun Y, et al. Effects of fish oil supplementation on kidney transplantation: a systematic review and
1621 meta-analysis of randomized, controlled trials. J Am Soc Nephrol 2005;16:2462e70. [17] Tamma SM, Shorter B, Toh KL, et al. Influence of polyunsaturated fatty acids on urologic inflammation. Int Urol Nephrol 2015;47:1753e61. [18] Câmara NO, Martins JO, Landgraf RG, Jancar S. Emerging roles for eicosanoids in renal diseases. Curr Opin Nephrol Hypertens 2009;18:21e7. [19] Butterly DW, Spurney RF, Ruiz P, et al. A role for leukotrienes in cyclosporine nephrotoxicity. Kidney Int 2000;57: 2586e93. [20] Brown SA, Brown CA, Crowell WA, et al. Effects of dietary polyunsaturated fatty acid supplementation in early renal insufficiency in dogs. J Lab Clin Med 2000;135:275e86. [21] Cristol JP, Vela C, Maggi MF, et al. Oxidative stress and lipid abnormalities in renal transplant recipients with or without chronic rejection. Transplantation 1998;65:1322e8. [22] Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957;226:497e509. [23] Armstrong KA, Hiremagalur B, Haluska BA, et al. Free fatty acids are associated with obesity, insulin resistance, and atherosclerosis in renal transplant recipients. Transplantation 2005;80:937e44. [24] Cofan F, Zambon D, Laguna JC, et al. Fatty acid composition of low-density lipoprotein in renal transplant recipients treated with cyclosporine. Transplant Proc 2002;34:374e6. [25] Lausada N, de Gómez Dumm INT, Georgina L, et al. Effect of different immunosuppressive therapies on the lipid pattern in kidney-transplanted rats. Transpl Int 2005;18:524e31. [26] Lausada N, de Gómez Dumm INT, Camihort G, et al. Lipid pattern in kidney-transplanted rats without immunosuppressive therapy. Transplant Proc 2002;34:380e3. [27] Oh JS, Kim SM, Sin YH, et al. Comparison of erythrocyte membrane fatty acid contents in renal transplant recipients and dialysis patients. Transplant Proc 2012;44:2932e5. [28] Alexander JW, Metze TJ, McIntosh MJ, et al. The influence of immunomodulatory diets on transplant success and complications. Transplantation 2005;79:460e5. [29] Lausada N, de Gómez Dumm INT, Raimondi JC, et al. Effect of cyclosporine and sirolimus on fatty acid desaturase activities in cultured HEPG2 cells. Transplant Proc 2009;41: 1865e70. [30] Wilusz M, Wasilewski G, Przybyłowski P, et al. Effect of immunosuppressive therapy on the serum fatty acids of phospholipids fraction in patients after heart transplantation. Transplant Proc 2014;46:2825e9. [31] Baggio B, Budakovic A, Ferraro A, et al. Relationship between plasma phospholipid polyunsaturated fatty acid composition and bone disease in renal transplantation. Transplantation 2005;80:1349e52. [32] Lauretani F, Semba RD, Bandinelli S, et al. Plasma polyunsaturated fatty acids and the decline of renal function. Clin Chem 2008;54:475e81. [33] Eide IA, Jenssen T, Hartmann A, et al. Plasma levels of marine n-3 polyunsaturated fatty acids and renal allograft survival. Nephrol Dial Transplant 2016;31:160e7. [34] Tayebi Khosroshahi H, Mousavi Toomatari SE, Akhavan Salamat S, et al. Effectiveness of omega-3 supplement on lipid profile and lipid peroxidation in kidney allograft recipients. Nephrourol Mon 2013;5:822e6. [35] Huang X, Stenvinkel P, Qureshi AR, et al. Essential polyunsaturated fatty acids, inflammation and mortality in dialysis patients. Nephrol Dial Transplant 2012;27:3615e20. [36] Naesens M, Kuypers DRJ, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clin J Am Soc Nephrol 2009;4:481e508. [37] Aldámiz-Echevarría L, Vallo A, Sanjurjo P, et al. Influence of diet on atherogenic risk in children with renal transplants. Pediatr Nephrol 2004;19:1039e45.
1622 [38] Toborek M, Lee YW, Garrido R, et al. Unsaturated fatty acids selectively induce an inflammatory environment in human endothelial cells. Am J Clin Nutr 2002;75: 119e25. [39] Kalogeropoulos N, Panagiotakos DB, Pitsavos C, et al. Unsaturated fatty acids are inversely associated and n-6/n-3 ratios are positively related to inflammation and coagulation markers in plasma of apparently healthy adults. Clin Chim Acta 2010;411: 584e91. [40] Poudel-Tandukar K, Nanri A, Matsushita Y, et al. Dietary intakes of alpha-linolenic and linoleic acids are inversely associated with serum C-reactive protein levels among Japanese men. Nutr Res 2009;29:363e70. [41] Laaksonen DE, Nyyssönen K, Niskanen L, et al. Prediction of cardiovascular mortality in middle-aged men by dietary and
WILUSZ, CIENIAWSKI, BUGAJSKA ET AL serum linoleic and polyunsaturated fatty acids. Arch Intern Med 2005;165:193e9. [42] Liu HQ, Qiu Y, Mu Y, et al. A high ratio of dietary n-3/n-6 polyunsaturated fatty acids improves obesity-linked inflammation and insulin resistance through suppressing activation of TLR4 in SD rats. Nutr Res 2013;33:849e58. [43] Brown SA, Finco DR, Brown CA. Is there a role for dietary polyunsaturated fatty acid supplementation in canine renal disease? J Nutr 1998;128(12 Suppl):2765Se7S. [44] Baggio B, Musacchio E, Priante G. Polyunsaturated fatty acids and renal fibrosis: pathophysiologic link and potential clinical implications. J Nephrol 2005;18:362e7. [45] Sieber J, Lindenmeyer MT, Kampe K, et al. Regulation of podocyte survival and endoplasmic reticulum stress by fatty acids. Am J Physiol Renal Physiol 2010;299:F821e9.
Department of Clinical Biochemistry, and bDepartment of Nephrology, Jagiellonian University Medical College, Cracow, Poland
ABSTRACT Background. Immunosuppressive medications often cause posttransplant hyperlipidemia. The effects of cyclosporine (CsA) and tacrolimus (Tac) on lipid profile is wellknown; however, there are very few studies related to the effect of these immunosuppressants on fatty acids (FA) of phosholipids fraction (PL) in renal transplant recipients (RTR). We sought to analyze the FA profile in PL fraction of RTR treated with Tac or CsA. Methods. The study included 65 renal transplant patients on CsA (n ¼ 24, group I) or Tac (n ¼ 41, group II), and 14 healthy controls. Individual serum FA concentrations were measured by gas chromatography. Chemstation software was used to analyze the data. Results. No differences between studied groups and controls were noted for monounsaturated FA, polyunsaturated n-3 FA (PUFA n-3), PUFA n-6, or the ratio of PUFA n-6 to PUFA n-3. The following mean values of FA were significantly higher in the CsA-RTR and Tac-RTR as compared with controls: total FA (P < .01 in both cases), saturated FA (SFA; P < .02 in both cases), C12 (P < .003 in both cases), C18 (P < .003 in both cases), and C18:2 (P < .01 for CsA RTR; P < .02 for Tac RTR). No differences between the measurements in patients on CsA and in patients on Tac were noticed. Significant correlation between SFA and eGFR was observed only in the CsA RTR group (P < .05). A negative relationship between PUFA n-6 and the estimated glomerular filtration rate was seen, but the correlation was not significant. Conclusions. Immunosuppressive drugs may affect FA metabolism, but the FA profile does not depend on the type of immunosuppressive drug administered.
P
ROGRESS in immunosuppressive management of patients after organ transplantation has significantly increased their overall survival. Tacrolimus (Tac) and cyclosporine (CsA) are among the most commonly used immunosuppressive drugs. These medications may, however, cause different side effects, among which lipid metabolic disorders are most frequently observed [1e4]. It has been shown that in patients receiving Tac the triglyceride (TG) levels are lower as compared with patients prescribed with CsA [3,5]. In contrast, treatment of the organ transplant patients with CsA causes significant increase of total cholesterol and low-density lipoprotein cholesterol levels [2,3,6]. Disturbances in lipid metabolism may also contribute to glomerular and interstitial injury that in turn
can promote renal disease progression. Posttransplant hyperlipidemia has been linked to cardiovascular disease, leading to a high morbidity and mortality rate [7,8]. Hyperlipidemia not only leads to development of atherosclerosis of coronary arteries, but also of renal artery, which can affect graft function [9,10]. Accumulation of lipids in nonadipose tissues may lead to cellular dysfunction and cell death, the phenomenon called lipotoxicity [11]. It has been confirmed that saturated fatty *Address correspondence to Małgorzata Wilusz, Department of Clinical Biochemistry, Jagiellonian University Medical College, University Children’s Hospital of Cracow, Wielicka 265, 30-663 Cracow, Poland. E-mail: [email protected]
0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.03.026
ª 2016 Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
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Transplantation Proceedings, 48, 1616e1622 (2016)
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acids (SFA), mainly palmitic acid, can be responsible for lipotoxicity of most cells, including renal cells (podocytes) [12]. Unlike SFA, it has been shown that monounsaturated fatty acids (MUFA) have protective effect on renal lipotoxicity, but the mechanism has not been fully understood yet. It is believed that the presence of oleic acid in podocytes can facilitate incorporation of palmitic acid into TG. Because of that lower level of intracellular concentration of palmitic acid is seen. On the other hand, increased rate of the b-oxidation of palmitic acid prevent accumulation of SFA in the cell [13,14]. Another class of fatty acids (FAs)dthe n-3 FAdmay protect kidney function by modulating the inflammatory response through downregulation of proinflammatory cytokines production, cyclooxygenase-2 activity, and an expression of endothelial leukocyte adhesion molecules [15e17]. In contrast, progressive deterioration of kidney function after dietary supplementation with n-6 FA has been shown [17]. Eicosanoids, which are the products of arachidonic acids (AAs) metabolism, have a wide range of biological action in inflammatory processes and immunity [18,19]. According to Brown et al. [20], the production of vasoactive eicosanoids may contribute to glomerular hyperfunction and consequently to glomerular damage. Dyslipidemia may also affect kidney function indirectly through vascular injury and oxidative stress. Cristol at al. [21] suggested that oxidation of polyunsaturated FAs (PUFA) may play a significant role in the vascular lesions. There are few studies related to the effect of different immunosuppressants on FA patterns in renal transplant recipients. Such findings would be significant, because FA play an important role in renal homeostasis and renal function. Furthermore, it may be speculated that changes in dietary habits towards higher intake of unsaturated FA could prevent or delay the progression of dyslipidemiarelated renal disease.
MATERIALS AND METHODS The study included 65 renal transplant recipients on immunosuppressive therapy: 24 patients treated with CsA (9 females, 15 males; mean age, 54 12 years [range, 25e65]) and 41 patients treated with Tac (15 females, 26 males; mean age, 51 13 years [range, 27e70]). All patients underwent renal transplantation, and were treated at the Department of Nephrology, Jagiellonian University Medical College, Cracow, Poland. The average time since transplantation was 42 months (range, 15e120). Therapeutic regimens were established individually for each patient, taking into account immunologic risk, comorbidity, ischemia time, and clinical experience. Safety and efficacy assessments were performed at scheduled study visits and therapy adjustments were introduced if necessary. Only patients who maintained their originally assigned calcineurin inhibitor were included in the study. The majority of patients received triple drug immunosuppressive therapy with a calcineurin inhibitor (CsA or Tac), mycophenolic acid, and glucocorticoids. Renal transplant recipients were treated with prednisone (in CsA group: 20 patients; in Tac group: 29 patients), methylprednisolone (in CsA group: 3 patients; in Tac group: 6 patients), and prednisolone (2 patients in Tac group). Five patients did not receive steroid therapy during the survey (1 in CsA group and 4 in Tac group). Each patient was 1 year posttransplant. Regardless of the calcineurin inhibitor used, all patients received low doses of steroids (average dose of glucocorticoids based on prednisone in CsA group, 6.66 mg; in Tac group, 5.19 mg). Furthermore, in patients of both groups glucocorticoids were used at comparable doses; thus, the effect of steroids was not taken into account in the statistical analysis. Patients with diabetes mellitus before renal transplantation, and the patients with a glomerular filtration rate (GFR) of <30 mL/min were excluded. The control group consisted of 14 healthy volunteers (3 males and 11 females; mean age, 48 12 years [range, 26e69]). The study protocol was approved by the Bioethics Committee of the Jagiellonian University and written informed consent was obtained from all patients. Baseline demographics and clinical characteristics of patients are presented in Table 1. Fasting blood samples for serum FA of PL determination were obtained from each patient. The blood serum was separated and
Table 1. Baseline Demographics and Clinical Characteristics of Patients Characteristic
Cyclosporine Group (n ¼ 24)
Tacrolimus Group (n ¼ 41)
Age range (y) Mean age (y) Male Female eGFR, mean (range), mL/min BMI, mean (range), kg/m2 Acute graft rejection Diabetes type II (after transplantation) Hypertension Lipid profile, mean (range), mmol/L TC TG LDL-C HDL-C Drug levels at the time of measurement, mean (range), ng/mL
25e65 54 12 15 9 65.3 (35.3e119.9) 26.7 (17.8e33.2) 2 8 8
27e70 51 13 26 15 75.4 (31.8e126.5) 26.8 (19.7e38.1) 3 8 15
4.97 (3.4e7.0) j 41.7%* 1.93 (0.53e3.3) j 33.3%* 2.95 (1.2e5.0) j 20.8%* 1.45 (0.7e2.1) j 12.5%* 452.00 (60.7e1167.5)
4.80 (3.0e6.9) j 43.9%* 1.89 (0.6e3.6) j 39.0%* 2.66 (1.5e4.1) j 21.9%* 1.32 (0.9e2.5) j 12.2%* 6.05 (3.9e11.5)
Abbreviations: BMI, body mass index; eGFR, estimated glomerular filtration rate; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides. *Percent of results above (TC 5 mmol/L; TG 1.7 mmol/L; LDL 3 mmol/L) or below (HDL <1 mmol/L for men, <1.2 mmol/L for women) recommended values.
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Table 2. Mean Values of Serum Fatty Acids of Phospholipids Fraction Concentration (mmol/L) in Renal Transplant Recipients Treated With Cyclosporine or Tacrolimus FA
SFA C12 C14 C16 C18 C24 MUFA C16:1 C18:1 PUFA C18:2 C20þC18:3(n-6) C18:3 C20:2 C20:4 C20:5 C22:6 PUFA n-3 PUFA n-6 PUFA n6/PUFA n3 SFA MUFA Total FA
Cyclosporine Group
3.92 17.51 1130.53 441.06 26.14
0.42† 0.94 31.17 18.61† 1.33
14.22 1.09 289.97 12.78 593.50 16.72 8.00 18.47 546.28 40.74 304.05 348.54 1175.00 3.56 1599.35 302.48 3463.61
25.98* 0.72 0.61 1.00 28.09 4.27 19.03 21.20 38.15 0.20 44.75** 11.76 93.52*
Tacrolimus Group
5.17 19.87 1117.93 436.29 27.02
0.39† 0.95 30.14 12.42† 1.07
18.10 1.46 297.37 12.05 594.04 15.94 8.33 19.21 561.25 52.07 297.73 343.04 1157.04 3.53 1564.38 318.03 3378.70
15.70** 0.66 0.43 0.61 18.54 3.09 13.43 40.53 25.67 0.17 38.27** 13.48 88.15*
Control Group
1.86 17.77 1011.09 359.40 27.10
0.27 2.40 65.31 21.23 1.83
19.80 4.80 289.54 32.54 503.80 15.23 8.33 18.48 523.72 51.55 260.70 315.87 1074.17 3.97 1388.26 293.86 2953.54
29.61 1.16 1.49 1.72 37.91 7.65 29.70 40.53 45.65 0.53 82.07 33.83 130.05
Values are presented as the mean SE (95% confidence interval). Abbreviations: FA, fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids. *P < .01; **P < .02; †P < .003 compared with the control group.
kept frozen at 70 C until measurement. The following analytical steps were taken: lipids extraction from serum with the use of Folsch method [22], separation of lipid fractions, and methylation of FA of PL fraction. Separation of the FA methyl esters was performed using gas chromatography equipped with flame ionization detector (Agilent Technologies 6890 Network GC System). The following FA were measured: myristic acid (C14), palmitic acid (C16), stearic acid (C18), palmitoleic acid (C16:1cis [n-7]), oleic acid (C18:1cis [n-9]), linoleic acid (LA; C18-2cis [n-6]), a-linolenic acid (ALA; C18:3cis [n-3]), 11,14-eicosadienoic acid (20:2cis [n-6]), AA (C20:4cis [n-6]), eicosapentaenoic acid (EPA; C20:5cis [n-3]), docosahexaenoic acid (DHA; C22:6cis [n-3]), and lignoceric acid (C24). Chemstation software was used to analyze the data. Total FA, total SFAs, total MUFAs, the sum of PUFA n-6, and the sum of PUFA n-3 concentrations, as well as the ratio of PUFA n-6 to PUFA n-3, were calculated. All results were expressed as mmol/L.
Statistical Analysis Descriptive statistics including mean values and standard error were calculated. To compare the FA profiles between patients on CsA or Tac and control group, one-way analysis of variance with a post hoc Newman-Keul test was applied. Pearson’s correlation coefficients were used to establish the relationship between FAs (PUFA n-6, PUFA n-3, total FA, SFA, and MUFA) and the estimated GFR (eGFR; calculated according to Cockcroft-Gault formula). The Student t test was performed to compare the differences in FA profiles between male and female patients. All analyses were done with the use of Statistica 10 (StatSoft). Statistical significance was set for P < .05.
RESULTS
The mean values (standard error) of individual FA of PL, as well as of total FAs, and the sum of: MUFA, SFA, PUFA n-3 and PUFA n-6, in renal transplant recipients receiving CsA or Tac and control group, are shown in Table 2. No differences between all measurements in patients on CsA and patients on Tac were observed. Patients treated with CsA and patients treated with Tac had significantly higher the mean SFA levels (P < .01 for CsA; P < .02 for Tac) as compared with controls. In both patient groups, the mean values of C12 and C18 were significantly higher than the mean value observed in control (P < .003 in both cases). The mean values of palmitic acid level were slightly higher in patient groups than in control; however, the differences were not significant. No differences between patient groups and controls were noted for C16:1 and C18:1, or for the sum of MUFA. Among PUFA, only the mean values of C18:2 were significantly higher in the CsA and Tac groups, as compared with control (P < .009 for CsA; P < .02 for Tac). The mean levels of C18:3, C20:2, C20:4, and C20:5 in both patient groups were similar to the values obtained in the control group. No differences between the studied groups and the control were noted for mean values of C22:6, PUFA n-3, and PUFA n-6; however, the highest values were obtained for the patient groups. The mean values of the ratio of PUFA n-6 to PUFA n-3 in the CsA and Tac groups and in control were similar. Total FA level was statistically higher in the transplanted patients than in the control group
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Fig 1. Relationship between polyunsaturated fatty acids (PUFA) n-6 concentration and estimated glomerular filtration rate (eGFR) in the cyclosporine (CsA; A) and tacrolimus (Tac; B) groups. Relationship between saturated fatty acid (SFA) concentration and eGFR in the CsA (C) and Tac (D) groups. Relationship between monounsaturated fatty acid (MUFA) concentration and eGFR in the CsA (E) and Tac (F) groups.
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(P < .01 for both groups). No differences in FA profile were observed between the male and female in CsA and Tac groups. No correlation between total FA concentration and eGFR in either patient group was noted. In addition, no correlation between PUFA n-3 and eGFR in the patient groups was observed. There was a negative relationship between PUFA n-6 and eGFR in CsA and Tac groups, but the correlations were not significant (Fig 1A, B). In contrast, significant correlation between SFA and eGFR in CsA group (Fig 1C) was observed, but not in the Tac group (Fig 1D). MUFA was related negatively in the CsA group (Fig 1E) and positively to eGFR in the Tac group (Fig 1F). However, the relationship was not significant. DISCUSSION
The different FA have distinct effects on cell’s metabolism and its function. It should be stressed that mainly SFA and their metabolites are responsible for induction of lipotoxicity [13,23]. In the present study, a higher level of SFA of PL in patients after renal transplantation was observed as compared with the control group thus confirming its possible role in podocytes lipotoxicity. In contrast, MUFA have protective effect on renal injury. Oleic acid can prevent SFA-induced lipotoxicity by facilitating incorporation of palmitic acid into TG. Furthermore, oleic acid stimulates palmitic acid b-oxidation leading to reducing the level of palmitic acid [13,14]. In our study, MUFA concentration in patients on immunosuppressive therapy matched the results in the control group. Similar findings were presented in the paper on MUFA content in low-density lipoprotein fraction in patients after renal transplantation [24] and in the research on rats with [25] and without [26] immunosuppressive therapy. In contrast, the level of oleic acid in erythrocyte membrane in kidney transplant recipients was lower, although palmitoleic acid and MUFA were significantly higher than in the control [27]. According to Alexander et al [28], supplementation with oleic acid was highly effective in longer allograft survival time in animals treated with CsA. There is little doubt that MUFA have protective role in renal transplant recipients. It is believed that immunosuppressive drugs such as CsA and Tac can influence the biosynthesis of different PUFA through modulation of desaturation and elongation processes, by decreasing D9 desaturase, and increasing D5 and D6 desaturase activity [29]. In contrast with our previous study performed in patients after heart transplantation [30], in the present study no differences in PUFA n-3 levels between patient and control groups were observed. The results are not in agreement with those of others [24,25,31]. It was demonstrated that PUFA n-3 reduces inflammation [32e34]; however, in some papers the association between PUFA n-3 and inflammation has not been confirmed [35]. It is known that PUFA n-6 have proinflommatory properties. AA, a substrate of eicosanoids synthesis, initiates the
WILUSZ, CIENIAWSKI, BUGAJSKA ET AL
inflammatory process. It is believed that CsA nephrotoxicity is associated with specific alterations in renal AA metabolism [36]. The role of AA metabolites in kidney dysfunction and acute rejection has been reported [19,25]. In the present study, the level of AA as well as the total PUFA n-6 were similar in patients and in the control groups. This is in agreement with the data obtained by Baggio et al. [31], Cofan et al. [24], and Oh et al. [27]. In contrast, a lower content of AA was noticed in children after renal transplantation [37] and in transplanted rats treated with different immunosuppressants [25]. In patients after renal transplantation, the depletion of LA in erythrocyte membrane and in low-density lipoprotein fraction was reported, although the differences were not significant. In the present study, significantly higher levels of LA in patient groups were observed. Based on recent findings, LA could have both proinflammatory [38] and antiinflammatory properties. Higher levels of LA correlates with decrease of IL-6 level [35,39] and is linked to a lower C-reactive protein concentration [40]. It was proved that an increase in circulating LA is associated with reduced inflammation and diminished cardiovascular risk [41]. The reported higher LA level in the present study seems to be beneficial for transplant recipients. The balance between PUFA n-6 and PUFA n-3 in the diet has changed substantially over the years. Currently, consumption of n-6 FA predominates. Because PUFA n-6 and n-3 compete for the same enzymes involved in FA desaturation and elongation, a higher n-6/n-3 ratio correlates positively with the level of proinflammatory cytokines [39]. Liu et al. [42] reported that increased PUFA n-3/n-6 ratio were associated with decreased serum TG and total cholesterol levels, improved insulin resistance and reduced proinflammatory cytokine production. The similar n-6/n-3 ratio in patient and in control groups observed in our study does not contradict the previous research because transplant patients are more cautious about their diet. The eGFR is accepted as the best overall measure of kidney function, and facilitates the detection, evaluation, and management of chronic kidney disease. An increased risk of complications is related directly to lower eGFR values. Recent epidemiological studies have demonstrated that serum PUFAs levels correlate with lower rates of creatinine clearance in an older population [32]. Our study showed that patients with low eGFR treated with CsA or Tac had higher PUFA n-6 concentrations, although the correlation was not significant. It could support the previous data on the effects of dietary PUFA n-6 supplementation on glomerular hemodynamic and renal injury, which showed that dietary supplementation with PUFA n-6 may hasten renal injury [20,43,44]. Additionally, it was observed that increased eGFR was accompanied by different MUFA levels, depending on the immunosuppressant used. In patients treated with CsA, the level of MUFA decreased, whereas in patients treated with Tac, MUFA concentration increased. The role of MUFA in the nephroprotection is documented [11,13,14]. However, the reason for different
SERUM FATTY ACIDS
effect of Tac and CsA on MUFA level is unknown. Similarly, the reason for difference in SFA level in relation to immunosuppressive therapy is unclear. A significant, negative correlation between eGFR and the level of SFA was only observed in patients treated with CsA. The toxicity of SFA on kidney function has already been described in literature [11,13,45]. In conclusion, it was confirmed that immunosuppressive drugs may affect FA metabolism in kidney transplant patients but the FA pattern did not depend on the type of immunosuppressive drug administered. The associations between the levels of PUFA n-6, MUFA, SFA, and eGFR indicate that FA play an important role in graft function in renal transplant patients. REFERENCES [1] Carta P, Zanazzi M, Di Maria L, et al. 5 year comparison of very low-dose cyclosporine and high-dose everolimus vs standard cyclosporine and enteric-coated mycophenolate in renal transplantation patients. Transplant Proc 2014;46:2228e30. [2] Ichimaru N, Takahara S, Kokado Y, et al. Changes in lipid metabolism and effect of simvastatin in renal transplant recipients induced by cyclosporine or tacrolimus. Atherosclerosis 2001;158: 417e23. [3] Colak T, Karakayali H, Yagmurdur MC, et al. Effect of conversion from cyclosporine to tacrolimus on lipid profiles in renal transplant recipients. Transplant Proc 2002;34:2081e2. [4] Mucha K, Foroncewicz B, Paczek L, et al. 36-month followup of 75 renal allograft recipients treated with steroids, tacrolimus, and azathioprine or mycophenolate mofetil. Transplant Proc 2003;35:2176e8. [5] Kanbay M, Yildirir A, Akcay A, et al. Effects of immunosuppressive drugs on serum lipid levels in renal transplant recipients. Transplant Proc 2006;38:502e5. [6] Akman B, Uyar M, Afsar B, et al. Lipid profile during azathioprine or mycophenolate mofetil combinations with cyclosporine and steroids. Transplant Proc 2007;39:135e7. [7] Sgambat K, He J, McCarter RJ, et al. Lipoprotein profile changes in children after renal transplantation in the modern immunosuppression era. Pediatr Transplant 2008;12:796e803. [8] Stoumpos S, Jardine AG, Mark PB. Cardiovascular morbidity and mortality after kidney transplantation. Transpl Int 2015;28: 10e21. [9] Massy ZA. Hyperlipidemia and cardiovascular disease after organ transplantation. Transplantation 2001;72(6 Suppl):S13e5. [10] Kimak E, Hałabis M, Baranowicz-Gaszczyk I. Relationships between serum lipid, lipoprotein, triglyceride-rich lipoprotein, and high-density lipoprotein particle concentrations in post-renal transplant patients. J Zhejiang Univ Sci B 2010;11:249e57. [11] Bobulescu IA. Renal lipid metabolism and lipotoxicity. Curr Opin Nephrol Hypertens 2010;19:393e402. [12] Kampe K, Sieber J, Orellana JM, et al. Susceptibility of podocytes to palmitic acid is regulated by fatty acid oxidation and inversely depends on acetyl-CoA carboxylases 1 and 2. Am J Physiol Renal Physiol 2014;306:F401e9. [13] Sieber J, Jehle AW. Free Fatty acids and their metabolism affect function and survival of podocytes. Front Endocrinol (Lausanne) 2014;5:186e7. [14] Listenberger LL, Han X, Lewis SE, et al. Triglyceride accumulation protects against fatty acid-induced lipotoxicity. Proc Natl Acad Sci U S A 2003;100:3077e82. [15] Donadio JV. n-3 Fatty acids and their role in nephrologic practice. Curr Opin Nephrol Hypertens 2001;10:639e42. [16] Tatsioni A, Chung M, Sun Y, et al. Effects of fish oil supplementation on kidney transplantation: a systematic review and
1621 meta-analysis of randomized, controlled trials. J Am Soc Nephrol 2005;16:2462e70. [17] Tamma SM, Shorter B, Toh KL, et al. Influence of polyunsaturated fatty acids on urologic inflammation. Int Urol Nephrol 2015;47:1753e61. [18] Câmara NO, Martins JO, Landgraf RG, Jancar S. Emerging roles for eicosanoids in renal diseases. Curr Opin Nephrol Hypertens 2009;18:21e7. [19] Butterly DW, Spurney RF, Ruiz P, et al. A role for leukotrienes in cyclosporine nephrotoxicity. Kidney Int 2000;57: 2586e93. [20] Brown SA, Brown CA, Crowell WA, et al. Effects of dietary polyunsaturated fatty acid supplementation in early renal insufficiency in dogs. J Lab Clin Med 2000;135:275e86. [21] Cristol JP, Vela C, Maggi MF, et al. Oxidative stress and lipid abnormalities in renal transplant recipients with or without chronic rejection. Transplantation 1998;65:1322e8. [22] Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957;226:497e509. [23] Armstrong KA, Hiremagalur B, Haluska BA, et al. Free fatty acids are associated with obesity, insulin resistance, and atherosclerosis in renal transplant recipients. Transplantation 2005;80:937e44. [24] Cofan F, Zambon D, Laguna JC, et al. Fatty acid composition of low-density lipoprotein in renal transplant recipients treated with cyclosporine. Transplant Proc 2002;34:374e6. [25] Lausada N, de Gómez Dumm INT, Georgina L, et al. Effect of different immunosuppressive therapies on the lipid pattern in kidney-transplanted rats. Transpl Int 2005;18:524e31. [26] Lausada N, de Gómez Dumm INT, Camihort G, et al. Lipid pattern in kidney-transplanted rats without immunosuppressive therapy. Transplant Proc 2002;34:380e3. [27] Oh JS, Kim SM, Sin YH, et al. Comparison of erythrocyte membrane fatty acid contents in renal transplant recipients and dialysis patients. Transplant Proc 2012;44:2932e5. [28] Alexander JW, Metze TJ, McIntosh MJ, et al. The influence of immunomodulatory diets on transplant success and complications. Transplantation 2005;79:460e5. [29] Lausada N, de Gómez Dumm INT, Raimondi JC, et al. Effect of cyclosporine and sirolimus on fatty acid desaturase activities in cultured HEPG2 cells. Transplant Proc 2009;41: 1865e70. [30] Wilusz M, Wasilewski G, Przybyłowski P, et al. Effect of immunosuppressive therapy on the serum fatty acids of phospholipids fraction in patients after heart transplantation. Transplant Proc 2014;46:2825e9. [31] Baggio B, Budakovic A, Ferraro A, et al. Relationship between plasma phospholipid polyunsaturated fatty acid composition and bone disease in renal transplantation. Transplantation 2005;80:1349e52. [32] Lauretani F, Semba RD, Bandinelli S, et al. Plasma polyunsaturated fatty acids and the decline of renal function. Clin Chem 2008;54:475e81. [33] Eide IA, Jenssen T, Hartmann A, et al. Plasma levels of marine n-3 polyunsaturated fatty acids and renal allograft survival. Nephrol Dial Transplant 2016;31:160e7. [34] Tayebi Khosroshahi H, Mousavi Toomatari SE, Akhavan Salamat S, et al. Effectiveness of omega-3 supplement on lipid profile and lipid peroxidation in kidney allograft recipients. Nephrourol Mon 2013;5:822e6. [35] Huang X, Stenvinkel P, Qureshi AR, et al. Essential polyunsaturated fatty acids, inflammation and mortality in dialysis patients. Nephrol Dial Transplant 2012;27:3615e20. [36] Naesens M, Kuypers DRJ, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clin J Am Soc Nephrol 2009;4:481e508. [37] Aldámiz-Echevarría L, Vallo A, Sanjurjo P, et al. Influence of diet on atherogenic risk in children with renal transplants. Pediatr Nephrol 2004;19:1039e45.
1622 [38] Toborek M, Lee YW, Garrido R, et al. Unsaturated fatty acids selectively induce an inflammatory environment in human endothelial cells. Am J Clin Nutr 2002;75: 119e25. [39] Kalogeropoulos N, Panagiotakos DB, Pitsavos C, et al. Unsaturated fatty acids are inversely associated and n-6/n-3 ratios are positively related to inflammation and coagulation markers in plasma of apparently healthy adults. Clin Chim Acta 2010;411: 584e91. [40] Poudel-Tandukar K, Nanri A, Matsushita Y, et al. Dietary intakes of alpha-linolenic and linoleic acids are inversely associated with serum C-reactive protein levels among Japanese men. Nutr Res 2009;29:363e70. [41] Laaksonen DE, Nyyssönen K, Niskanen L, et al. Prediction of cardiovascular mortality in middle-aged men by dietary and
WILUSZ, CIENIAWSKI, BUGAJSKA ET AL serum linoleic and polyunsaturated fatty acids. Arch Intern Med 2005;165:193e9. [42] Liu HQ, Qiu Y, Mu Y, et al. A high ratio of dietary n-3/n-6 polyunsaturated fatty acids improves obesity-linked inflammation and insulin resistance through suppressing activation of TLR4 in SD rats. Nutr Res 2013;33:849e58. [43] Brown SA, Finco DR, Brown CA. Is there a role for dietary polyunsaturated fatty acid supplementation in canine renal disease? J Nutr 1998;128(12 Suppl):2765Se7S. [44] Baggio B, Musacchio E, Priante G. Polyunsaturated fatty acids and renal fibrosis: pathophysiologic link and potential clinical implications. J Nephrol 2005;18:362e7. [45] Sieber J, Lindenmeyer MT, Kampe K, et al. Regulation of podocyte survival and endoplasmic reticulum stress by fatty acids. Am J Physiol Renal Physiol 2010;299:F821e9.