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F2-Isoprostanes in Kidney Transplant Patients: Relationship With Inflammatory Markers
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F2-Isoprostanes in Kidney Transplant Patients: Relationship With Inflammatory Markers R. Lauzurica, M.C. Pastor, B. Bayés, J.M. Hernández, J. Bonet, M.A. Llopis, L. Carrera, and R. Romero ABSTRACT This prospective study evaluated the relationship between inflammation and oxidative stress in a group of dialysis patients just before and 3 months after kidney transplantation and compared the results with a control group of healthy subjects. The oxidative stress markers determined were different F2-isoprostane isomers. The inflammatory markers included C-reactive protein, interleukin-6, tumor necrosis factor-␣, and pregnancy-associated plasma protein A. Forty-three patients were the study group and 50 healthy subjects from a hospital blood bank as controls. The results showed levels of inflammatory and oxidative stress markers to be higher in the dialysis patients than in the control group, although they improved following kidney transplantation. Finally, significant correlations were observed between F2-isoprostane isomers and inflammatory markers.
O
XIDATIVE stress and inflammation are closely interrelated features. Oxidative stress and inflammation are increased in patients with chronic kidney insufficiency, clearly contributing factors to cardiovascular complications.1,2 Reactive oxygen species (ROS) are extremely unstable and highly reactive metabolites, which makes them difficult to detect in vivo. The oxidative change in humans is measured by different biomolecules such as lipids, proteins, carbohydrates, nucleic acids, etc. Increased lipid peroxidation products such as oxidized LDL, malondialdehyde, isolevuglandins, and F2-isoprostanes (F2-IPs) have been detected in patients with chronic kidney impairment.1,2 The F2 prostaglandin family isomers are products of the attack of free radicals on cellular membrane phospholipids or circulating LDL. These isomers are one of the best in vivo oxidative stress markers.3 The aim of this study was to determine the level of F2-IPs in patients on dialysis just prior to kidney transplantation, their evolution 3 months after transplantation, and their relationship with different inflammatory markers.
PATIENTS AND METHODS This prospective study included 43 kidney transplant patients (32 men/11 women; mean age 54.9 ⫾ 11.8 years [range: 26 –71 years]). The following parameters were determined just before and 3 months after transplantation, when patients were in a clinically stable condition: C-reactive protein (CRP) by nephelometry in a BNP-Prospect apparatus (Behring); tumor necrosis factor-␣ (TNF-␣) and interleukin6 (IL-6) by immunometric chemoluminescence (DPC); and ultrasensitive pregnancy-associated plasma protein A (PAPP-A) by ELISA (DSL). The F2-IPs were assessed with mass spectrometry with negative chemical ionization, using isotopic dilution with d4-F2␣. QuantiFrom the Servicios de Nefrología y Bioquimica (R.L., B.B., J.B.), Hospital Universitario Germans Trias i Pujol (M.C.P., J.M.H., M.A.L., L.C., R.R.), Badalona, Spain. Supported by Fondo para Investigación, Sanitaria del Ministerio de Salud (FIS: 021392). Address reprint requests to Dr. Ricardo Lauzurica, Kidney Transplantation Unit, Hospital Universitari Germans Trias i Pujol, Ctra. Del Canyet, s/n, 08916 Badalona, Spain. E-mail:rlauzu@ ns.hugtip.scs.es
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.106
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3842
Transplantation Proceedings, 37, 3842–3843 (2005)
INFLAMMATORY MARKERS fication of the following isomers was performed using the aforementioned method: F2--IP, 8-epi-F2␣-IP, 11--F2-␣-IP, and F2-␣-IP. Fifty healthy individuals from the hospital blood bank, matched by age and gender with the study group, were used as controls.
Statistics All results were expressed as mean values ⫾ SD with P ⬍ .05 considered statistically significant in each case. The Mann-Whitney U test was used for skewed variables. To test for associations among the results, Pearson correlation coefficients were calculated between F2-IPs and the inflammatory markers.
RESULTS
The four F2-IP isomers (pg/mL) were increased in patients on dialysis and at the time of transplantation compared with the control group: F2--IP (63.36 ⫾ 42.5 vs 81.57 ⫾ 39.7 P ⫽ .042); 8-epi-F2-␣-IP (74.43 ⫾ 32.2 vs 112.2 ⫾ 54.9; P ⫽ .007); 11--F2-␣-IP (248.55 ⫾ 104.6 vs 309.63 ⫾ 188.12; P ⫽ .473); F2-␣-IP (344.82 ⫾ 111.27 vs 522.57 ⫾ 274.6; P ⫽ .002). Three months after transplantation, an improvement occurred in F2-IP values: 8-epi-F2-␣-IP (112.2 ⫾ 54.9 vs 79.05 ⫾ 32.1; P ⫽ .05) and F2-␣-IP (522.57 ⫾ 274.6 vs 360.35 ⫾ 144; P ⫽ .046). Likewise, an increase occurred in the inflammatory markers in pretransplant patients on dialysis compared with the control group. With the exception of IL-6, these markers decreased significantly 3 months after transplantation: CRP (mg/L) 3.3 ⫾ 3.9 (controls) vs 11.15 ⫾ 20.5 (dialysis) P ⫽ .021 vs 7.54 ⫾ 10.8 (3 months after transplantation) P ⫽ NS; IL-6 (pg/mL) 2.25 ⫾ 1.31 vs 4.98 ⫾ 3.13 (P ⫽ .0001) vs 8.02 ⫾ 15.37; TNF-␣ (pg/mL) 4.58 ⫾ 1.12 vs 15.28 ⫾ 14.88 (P ⫽ .0001) vs 11.3 ⫾ 4.83 (P ⫽ .08); PAPP-A (mUI/L) 0.98 ⫾ 0.46 vs 2.18 ⫾ 1.39 (P ⫽ .0001) vs 1.87 ⫾ 1.79 (P ⫽ .078). Despite these posttransplant improvements, inflammatory markers were still higher than those in healthy controls. Only 6 of the 43 patients in the study group are at the 1-year mark posttransplant. In these cases, the inflammatory markers are still decreasing: CRP: 2.39 ⫾ 1.55 (mg/L); IL-6: 3.06 ⫾ 0.85 (pg/mL); TNF-␣: 7.68 ⫾ 3.33 (pg/mL); PAPP-A:1.42 ⫾ 0.98 (mUI/L). Finally, we found a correlation between the F2-IP isomers and the inflammatory markers (Table 1). DISCUSSION
The potential synergy between inflammation and oxidative stress is one of the most widely recognized hypotheses explaining the development of accelerated atherosclerosis in patients with renal insufficiency. The F2-IPs are isomers of F2 prostaglandins that form during the process of LDL oxidation. They are considered to be excellent markers of in vivo oxidative stress. There have been several studies on oxidative stress in patients with chronic renal insufficiency on dialysis. However, few of those studies have prospectively analyzed this
3843 Table 1. Correlation Between F2-Isoprostanes Versus Inflammatory Markers
CRP P IL-6 P TNF-␣ P PAPP-A P
F2--IP
8-epi-F2-␣-IP
11-F2-␣-IP
F2␣-IP
0.052 NS 0.349 0.002 0.610 0.0001 0.393 0.003
⫺0.139 NS 0.282 0.012 0.522 0.0001 0.382 0.004
⫺0.067 NS 0.307 0.006 0.330 0.003 0.211 NS
0.061 NS 0.0369 0.0001 0.492 0.0001 0.291 0.029
Abbreviations: F2--IP, 8-epi-F2-␣-IP, 11-F2-␣-IP, F2␣-IP, F2-isoprostane isomers; CRP, C-reactive protein; IL-6, interleukin-6; TNF-␣, tumor necrosis factor-␣; PAPP-A, pregnancy-associated plasma protein-A.
complication in kidney transplant patients in relation to oxidative stress and inflammation. Our study shows several important points. First, patients on hemodialysis just prior to kidney transplantation, and therefore in a clinically stable condition (absence of any infectious and/or acute or chronic inflammatory episode), showed an increase in all analyzed F2-IP isomers compared with controls. This observation has been previously described,4 – 6 using total F2-IP values. However, we are not aware of any other studies showing improvement in oxidative stress 3 months after transplantation, as assessed by a decrease in all F2-IP isomer values. Second, the increased inflammation in patients on dialysis, including PAPP-A, similar to F2-IP improves after transplantation, despite not reaching a level considered to be normal. Finally, there was a relationship between oxidative stress and inflammation: F2-IPs correlated with most inflammatory markers, independent of circulating lipids. However, it is noteworthy that, according to our results, all of the isomers correlated with CRP, the standard inflammatory marker, and the one most routinely described in relation to F2-IP.4 – 6 These results show alterations and relationships between oxidative stress and inflammation in patients with chronic renal insufficiency on dialysis with short-term improvement after transplantation. REFERENCES 1. Vaziri ND: Oxidative stress in uremia: nature, mechanisms, and potential consequences. Semin Nephrol 24:469, 2004 2. Himmelfarb J, Stenvinkel P, Ikizler TA, et al: The elephant in uremia: oxidant stress as a unifying concept of cardiovascular disease in uremia. Kidney Int 62:1524, 2002 3. Lawson JA, Rokach J, Fitzgerald GA: Isoprostanes formation, analysis and use as indices of lipid peroxidation in vivo. J Biol Chem 274:24441, 1999 4. Spittle MA, Hoenich NA, Handelman GJ, et al: Oxidative stress and inflammation in hemodialysis patients. Am J Kidney Dis 38:1408, 2001 5. Handelman GJ, Walter MF, Adhikarla R, et al: Elevated plasma F2-isoprostanes in patients on long-term hemodialysis. Kidney Int 59:1960, 2001 6. Ikizler TA, Morrow JA, Roberts LJ, et al: Clin Nephrol 58:190, 2002
F2-Isoprostanes in Kidney Transplant Patients: Relationship With Inflammatory Markers R. Lauzurica, M.C. Pastor, B. Bayés, J.M. Hernández, J. Bonet, M.A. Llopis, L. Carrera, and R. Romero ABSTRACT This prospective study evaluated the relationship between inflammation and oxidative stress in a group of dialysis patients just before and 3 months after kidney transplantation and compared the results with a control group of healthy subjects. The oxidative stress markers determined were different F2-isoprostane isomers. The inflammatory markers included C-reactive protein, interleukin-6, tumor necrosis factor-␣, and pregnancy-associated plasma protein A. Forty-three patients were the study group and 50 healthy subjects from a hospital blood bank as controls. The results showed levels of inflammatory and oxidative stress markers to be higher in the dialysis patients than in the control group, although they improved following kidney transplantation. Finally, significant correlations were observed between F2-isoprostane isomers and inflammatory markers.
O
XIDATIVE stress and inflammation are closely interrelated features. Oxidative stress and inflammation are increased in patients with chronic kidney insufficiency, clearly contributing factors to cardiovascular complications.1,2 Reactive oxygen species (ROS) are extremely unstable and highly reactive metabolites, which makes them difficult to detect in vivo. The oxidative change in humans is measured by different biomolecules such as lipids, proteins, carbohydrates, nucleic acids, etc. Increased lipid peroxidation products such as oxidized LDL, malondialdehyde, isolevuglandins, and F2-isoprostanes (F2-IPs) have been detected in patients with chronic kidney impairment.1,2 The F2 prostaglandin family isomers are products of the attack of free radicals on cellular membrane phospholipids or circulating LDL. These isomers are one of the best in vivo oxidative stress markers.3 The aim of this study was to determine the level of F2-IPs in patients on dialysis just prior to kidney transplantation, their evolution 3 months after transplantation, and their relationship with different inflammatory markers.
PATIENTS AND METHODS This prospective study included 43 kidney transplant patients (32 men/11 women; mean age 54.9 ⫾ 11.8 years [range: 26 –71 years]). The following parameters were determined just before and 3 months after transplantation, when patients were in a clinically stable condition: C-reactive protein (CRP) by nephelometry in a BNP-Prospect apparatus (Behring); tumor necrosis factor-␣ (TNF-␣) and interleukin6 (IL-6) by immunometric chemoluminescence (DPC); and ultrasensitive pregnancy-associated plasma protein A (PAPP-A) by ELISA (DSL). The F2-IPs were assessed with mass spectrometry with negative chemical ionization, using isotopic dilution with d4-F2␣. QuantiFrom the Servicios de Nefrología y Bioquimica (R.L., B.B., J.B.), Hospital Universitario Germans Trias i Pujol (M.C.P., J.M.H., M.A.L., L.C., R.R.), Badalona, Spain. Supported by Fondo para Investigación, Sanitaria del Ministerio de Salud (FIS: 021392). Address reprint requests to Dr. Ricardo Lauzurica, Kidney Transplantation Unit, Hospital Universitari Germans Trias i Pujol, Ctra. Del Canyet, s/n, 08916 Badalona, Spain. E-mail:rlauzu@ ns.hugtip.scs.es
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.106
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3842
Transplantation Proceedings, 37, 3842–3843 (2005)
INFLAMMATORY MARKERS fication of the following isomers was performed using the aforementioned method: F2--IP, 8-epi-F2␣-IP, 11--F2-␣-IP, and F2-␣-IP. Fifty healthy individuals from the hospital blood bank, matched by age and gender with the study group, were used as controls.
Statistics All results were expressed as mean values ⫾ SD with P ⬍ .05 considered statistically significant in each case. The Mann-Whitney U test was used for skewed variables. To test for associations among the results, Pearson correlation coefficients were calculated between F2-IPs and the inflammatory markers.
RESULTS
The four F2-IP isomers (pg/mL) were increased in patients on dialysis and at the time of transplantation compared with the control group: F2--IP (63.36 ⫾ 42.5 vs 81.57 ⫾ 39.7 P ⫽ .042); 8-epi-F2-␣-IP (74.43 ⫾ 32.2 vs 112.2 ⫾ 54.9; P ⫽ .007); 11--F2-␣-IP (248.55 ⫾ 104.6 vs 309.63 ⫾ 188.12; P ⫽ .473); F2-␣-IP (344.82 ⫾ 111.27 vs 522.57 ⫾ 274.6; P ⫽ .002). Three months after transplantation, an improvement occurred in F2-IP values: 8-epi-F2-␣-IP (112.2 ⫾ 54.9 vs 79.05 ⫾ 32.1; P ⫽ .05) and F2-␣-IP (522.57 ⫾ 274.6 vs 360.35 ⫾ 144; P ⫽ .046). Likewise, an increase occurred in the inflammatory markers in pretransplant patients on dialysis compared with the control group. With the exception of IL-6, these markers decreased significantly 3 months after transplantation: CRP (mg/L) 3.3 ⫾ 3.9 (controls) vs 11.15 ⫾ 20.5 (dialysis) P ⫽ .021 vs 7.54 ⫾ 10.8 (3 months after transplantation) P ⫽ NS; IL-6 (pg/mL) 2.25 ⫾ 1.31 vs 4.98 ⫾ 3.13 (P ⫽ .0001) vs 8.02 ⫾ 15.37; TNF-␣ (pg/mL) 4.58 ⫾ 1.12 vs 15.28 ⫾ 14.88 (P ⫽ .0001) vs 11.3 ⫾ 4.83 (P ⫽ .08); PAPP-A (mUI/L) 0.98 ⫾ 0.46 vs 2.18 ⫾ 1.39 (P ⫽ .0001) vs 1.87 ⫾ 1.79 (P ⫽ .078). Despite these posttransplant improvements, inflammatory markers were still higher than those in healthy controls. Only 6 of the 43 patients in the study group are at the 1-year mark posttransplant. In these cases, the inflammatory markers are still decreasing: CRP: 2.39 ⫾ 1.55 (mg/L); IL-6: 3.06 ⫾ 0.85 (pg/mL); TNF-␣: 7.68 ⫾ 3.33 (pg/mL); PAPP-A:1.42 ⫾ 0.98 (mUI/L). Finally, we found a correlation between the F2-IP isomers and the inflammatory markers (Table 1). DISCUSSION
The potential synergy between inflammation and oxidative stress is one of the most widely recognized hypotheses explaining the development of accelerated atherosclerosis in patients with renal insufficiency. The F2-IPs are isomers of F2 prostaglandins that form during the process of LDL oxidation. They are considered to be excellent markers of in vivo oxidative stress. There have been several studies on oxidative stress in patients with chronic renal insufficiency on dialysis. However, few of those studies have prospectively analyzed this
3843 Table 1. Correlation Between F2-Isoprostanes Versus Inflammatory Markers
CRP P IL-6 P TNF-␣ P PAPP-A P
F2--IP
8-epi-F2-␣-IP
11-F2-␣-IP
F2␣-IP
0.052 NS 0.349 0.002 0.610 0.0001 0.393 0.003
⫺0.139 NS 0.282 0.012 0.522 0.0001 0.382 0.004
⫺0.067 NS 0.307 0.006 0.330 0.003 0.211 NS
0.061 NS 0.0369 0.0001 0.492 0.0001 0.291 0.029
Abbreviations: F2--IP, 8-epi-F2-␣-IP, 11-F2-␣-IP, F2␣-IP, F2-isoprostane isomers; CRP, C-reactive protein; IL-6, interleukin-6; TNF-␣, tumor necrosis factor-␣; PAPP-A, pregnancy-associated plasma protein-A.
complication in kidney transplant patients in relation to oxidative stress and inflammation. Our study shows several important points. First, patients on hemodialysis just prior to kidney transplantation, and therefore in a clinically stable condition (absence of any infectious and/or acute or chronic inflammatory episode), showed an increase in all analyzed F2-IP isomers compared with controls. This observation has been previously described,4 – 6 using total F2-IP values. However, we are not aware of any other studies showing improvement in oxidative stress 3 months after transplantation, as assessed by a decrease in all F2-IP isomer values. Second, the increased inflammation in patients on dialysis, including PAPP-A, similar to F2-IP improves after transplantation, despite not reaching a level considered to be normal. Finally, there was a relationship between oxidative stress and inflammation: F2-IPs correlated with most inflammatory markers, independent of circulating lipids. However, it is noteworthy that, according to our results, all of the isomers correlated with CRP, the standard inflammatory marker, and the one most routinely described in relation to F2-IP.4 – 6 These results show alterations and relationships between oxidative stress and inflammation in patients with chronic renal insufficiency on dialysis with short-term improvement after transplantation. REFERENCES 1. Vaziri ND: Oxidative stress in uremia: nature, mechanisms, and potential consequences. Semin Nephrol 24:469, 2004 2. Himmelfarb J, Stenvinkel P, Ikizler TA, et al: The elephant in uremia: oxidant stress as a unifying concept of cardiovascular disease in uremia. Kidney Int 62:1524, 2002 3. Lawson JA, Rokach J, Fitzgerald GA: Isoprostanes formation, analysis and use as indices of lipid peroxidation in vivo. J Biol Chem 274:24441, 1999 4. Spittle MA, Hoenich NA, Handelman GJ, et al: Oxidative stress and inflammation in hemodialysis patients. Am J Kidney Dis 38:1408, 2001 5. Handelman GJ, Walter MF, Adhikarla R, et al: Elevated plasma F2-isoprostanes in patients on long-term hemodialysis. Kidney Int 59:1960, 2001 6. Ikizler TA, Morrow JA, Roberts LJ, et al: Clin Nephrol 58:190, 2002