- Email: [email protected]
Detection of Citrate Synthase Autoantibodies in Rats with Chronic Allograft Nephropathy
Detection of Citrate Synthase Autoantibodies in Rats with Chronic Allograft Nephropathy L.Y. Zhang, Y.P. Lu, L. Yang, G.H. Luo, J. Song, and Y.P. Li ABSTRACT Citrate synthase (CS) is the one of the key enzymes in the citric acid cycle and an important mitochondrial autoantigen. The autoimmune responses against CS have not been studied in chronic allograft nephropathy (CAN). This study investigated the role of specific CS autoantibodies in rats bearing renal allografts affected with CAN. Methods. Fisher344 rat renal grafts were orthotopically transplanted into Lewis rats following the procedure of Kamada with our modification. Lewis-to-Lewis and Fisher344to-Fisher344 kidney transplantations were also performed as autologous control groups (each n ⫽ 9). All the allograft recipients given cyclosporine (10 mg/kg⫺1d⫺1 ⫻ 10 d) were divided into four groups (each n ⫽ 9): (1) vehicle: normal saline orally; (2) cyclosporine: 6 mg/kg⫺1d⫺1; (3)FK506: 0.15 mg/kg⫺1d⫺1; (4) mycophenolate mofetil (MMF): 20 mg/ kg⫺1d⫺1. At 4, 8, and 12 weeks posttransplantation, the animals were sacrificed to harvest sera and renal allografts. The serum creatinine (SCr) was measured and pathological changes assessed according to Banff 97 criteria. IgM and IgG isotypes of CS antibodies were detected in all recipient sera by enzyme linked immunosorbent assays. Results. Both IgM and IgG isotype CS autoantibodies were observed in the sera of all the recipients before and after transplantation, but the levels of IgM CS autoantibody were obviously higher than IgG isotype in all the blood samples. It was stable not only in autologous but also in allograft groups. In both autologous groups, the SCr and IgM and IgG isotype CS autoantibodies showed no obvious change before and after transplantation, and no typical CAN occurred. The values of IgG isotype of CS autoantibody (⌬OD) at 4, 8 and 12 weeks were stable. At 4 weeks, the values of SCr, Banff score, and IgG isotype CS autoantibody (⌬OD) were not significantly different (P ⬎ .05) among the allograft groups. At 8 and 12 weeks, with progression of CAN in vehicle, cyclosporine and FK506 groups’ values of SCr, Banff score, and IgG (⌬OD) also increased dramatically (P ⫽ .005) in all three groups when compared with the baseline and 4 week values, but the differences among the three groups were not significant (P ⬎ .05). At 8 and 12 weeks, the MMF group suffered mild-to-moderate CAN, but the values of SCr and Banff score were significantly lower than those in the other three groups. MMF significantly inhibited the formation of IgG (⌬OD) when compared with the other three groups (P ⫽ .02). Conclusion. This study suggested that the IgG isotype of CS autoantibody contributes to CAN after kidney transplantation. The IgM isotype is physiological. MMF significantly
From the Transplantation Institute, West China Hospital, Chengdu, Sichuan, China. This study was supported by a grant from the Chinese National Basic Science Foundation (2003CB515504), a grant from the Doctor-Foundation (JYB00402051025), the Program for Changjiang Scholars and Innovative Research Team in
University, Chinese Educational Ministry, and a grant from Shanghai Roche Pharmaceutics Ltd. Address reprint requests to Prof Yi Ping Lu, Transplantation Institute, West China Hospital, Sichuan University, Guoxuexiang 37, Chengdu, China 610041. E-mail: [email protected]
0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.08.071
© 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
4366
Transplantation Proceedings, 41, 4366 – 4368 (2009)
CITRATE SYNTHASE AUTOANTIBODIES
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inhibited the formation of IgG isotype CS autoantibody, which may be related to its effects to alleviate CAN. HRONIC ALLOGRAFT NEPHROPATHY (CAN) is one of the major causes of late renal graft loss.1,2 The prevalence of CAN is as high as 60% to 70% on protocol biopsies after the first year.3,4 However, the complex causes of CAN, which involve immunological and nonimmunological factors, have not been fully elucidated. Citrate synthase (CS) is a key enzyme in the citric acid cycle. It plays a central role in the regulation of energy homeostasis and cell metabolism; meanwhile, it is an important mitochondrial autoantigen. In this study, we sought to investigate the role of CS-specific autoantibodies in rat renal allografts displaying CAN.
C
MATERIALS AND METHODS Animals Male inbred Fisher (F344; RT1lv1) and Lewis (LEW; RT11) rats weighing 250 to 300 g were obtained from the Experimental Animal Center of Chinese Academy of Science (Shanghai, China). The animals had free access to water and rat chow under standard conditions.
Kidney Transplantation and Experimental Groups Kidney transplantations were performed from F344 to Lewis (allogeneic), Lewis to Lewis and Fisher 344 to Fisher 344 rats (autologous, each n ⫽ 9) following the procedure of Kamada5 with our modification.6 After allograft recipients were first treated with cyclosporine (CsA: 10 mg/ kg⫺1d⫺1 ⫻ 10 d), they were divided into four groups (each n ⫽ 9): (1)vehicle: vehicle orally; (2)CsA: 6 mg/kg⫺1d⫺1; (3)FK506: 0.15 mg/kg⫺1d⫺1; (4) mycophenolate mofetil (MMF): 20 mg/kg⫺1d⫺1. At 4, 8, and 12 weeks posttransplant, the hosts were sacrificed to harvest sera and renal allografts. The serum creatinine (SCr) was measured and pathological changes assessed according to the Banff 97 criteria.
Assessment of Renal Morphology Formalin-fixed, paraffin-embedded sections of renal tissue samples were stained with periodic acid-Schiff, hematoxylin-eosin, and Masson’s trichrome. All sections were scored based on pathological changes in glomerular, tubular, and vascular tissues upon light microscopy. CAN was determined by two independent nephropathologists according to the Banff 97 criteria.
Detection of Anti-Citrate Synthase Autoantibodies IgM and IgG isotype CS autoantibodies were detected by a binding, indirect, enzyme-linked immunosorbent assay. Citrate synthase from porcine heart (Sigma) was coated (2.5 g/well) overnight at 4 °C, which was followed by a 1-hour incubation at 37 °C on 96-well polystyrol plates (NUNC, Denmark). The plates were blocked using 20% bovine serum albumin in carbonate-buffered saline at 4 °C for 2 hours. Serum samples diluted 1:50 were incubated for 1 hour; before the plates were incubated for 60 minutes with secondary antibodies of rabbit anti-rat IgG horseradish peroxidase (HRPO) or rabbit anti-rat IgM horseradish peroxidase (Sigma). The color reaction using ortho-phenylenediamine
(Sigma) was measured on a Thermo Multiskan MK3 (USA) microplate reader at 450 nm. Results were expressed as OD: namely, the OD from a postoperative sample minus the OD obtained in a reference preoperative sample from the same rat that had been obtained 1 week before the operation.
Statistical Analysis All data shown as mean values ⫾ SD were analyzed by Student’s t tests with comparisons between groups also performed by one-way analysis of variance. P ⬍ .05 was considered significant. We employed SPSS13.0 statistical software.
RESULTS Changes in the Renal Graft Function
Renal allograft function in autologous groups was stable, namely, SCr values (mol/L) of 67.67 ⫾ 6.43, 67.76 ⫾ 4.14, and 70.99 ⫾ 8.19 (Lewis) and 71.23 ⫾ 5.37, 69.13 ⫾ 4.26, and 70.42 ⫾ 7.78 (Fisher344) at 4, 8, and 12 weeks, respectively (P ⬎ .9). At 4 weeks, the SCr in vehicle, CsA, FK506, and MMF groups were 110.97 ⫾ 55.83, 75.27 ⫾ 8.91, 86.07 ⫾ 18.13, and 72.41 ⫾ 7.04, respectively (P ⬎ .05). The SCr obviously increased at 8 weeks and the hosts became uremic at 12 weeks, namely, SCr levels of 275.23 ⫾ 23.51, 295.26 ⫾ 32.07, and 263.64 ⫾ 50.97 at 8; and 516.78 ⫾ 31.59, 525.18 ⫾ 33.67, and 476.93 ⫾ 61.63 at 12 weeks in vehicle, CsA, and FK506 groups, respectively. The SCr values in the MMF group were 138.26 ⫾ 28.48 and 305.94 ⫾ 32.92 at 8 and 12 weeks, respectively, which were significantly lower than those in other three groups (P ⬍ .01). Changes in the Renal Graft Morphology
There were no signs of acute rejection in any group. There was no evidence of CAN in the autologous groups. The Banff scores were 0.33 ⫾ 0.58, 0.67 ⫾ 0.58, and 1.33 ⫾ 0.26 (Lewis) (P ⬎ .05); 0.27 ⫾ 0.58, 0.71 ⫾ 0.45, and 2.33 ⫾ 0.58 (Fisher 344) at 4, 8, and 12 weeks, respectively. At 4 weeks the Banff scores in the vehicle, CsA, FK506, and MMF groups were 1.67 ⫾ 0.58, 1.33 ⫾ 0.58, 1.00 ⫾ 1.00, and 0.67 ⫾ 0.58, respectively. The differences were not significant (P ⬎ .05). The typical pathologic changes of CAN, including vascular and glomerular sclerosis as well as interstitial fibrosis, appeared at 8 weeks in vehicle, CsA, and FK506 groups. At 12 weeks, segmental or global glomerulosclerosis was noted in association with increasing tubular atrophy, interstitial fibrosis, intimal proliferation, and gradual luminal occlusion of the cortical vessels in the vehicle, CsA, and FK506 groups, namely, Banff scores of 5.33 ⫾ 0.58, 12.67 ⫾ 1.16; 4.67 ⫾ 1.53 and 13.33 ⫾ 1.53; 5.67 ⫾ 0.58 and 12.33 ⫾ 3.79 at 8 and 12 weeks, respectively. At 8 and 12 weeks the Banff scores of the MMF group were 1.67 ⫾ 1.16 and 6.67 ⫾ 1.53, respectively, which were significantly lower than those of other three allograft groups (P ⬍ .05).
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Anticitrate Synthase Autoantibodies
Both IgM and IgG isotype CS autoantibodies were detected in the sera of all recipients before and after transplantation, but the levels of IgM CS autoantibody were obviously higher than those of the IgG isotype. After transplantation they were stable not only in the autologous groups, but also in the allograft groups. At 4 weeks the differences in the values of IgG isotype CS autoantibody (⌬OD) in vehicle, CsA, FK506, and MMF groups were not significant (P ⬎ .05). With progression of CAN at 8 and 12 weeks, the values of IgG (⌬OD) also increased dramatically in vehicle, CsA, and FK506 groups (P ⫽ .005), but the differences were not significant (P ⬎ .05). The levels of IgG (⌬OD) in the MMF group were significantly lower than those in the other three groups (P ⫽ .02; Table 1). DISCUSSION
Despite improvements in immunosuppression, CAN still causes most kidney allograft losses and remains the central clinical challenge.7,8 In spite of the development of sensitive anti-HLA screening assays and crossmatching techniques, there are antibody-mediated rejection episodes as confirmed by C4d deposition, suggesting a role for non-HLA antibodies.9 The non-HLA antibodies associated with solid organ transplantation are a variety of autoantibodies commonly found in autoimmune diseases,10 such as anti-nuclear, anti-nucleoprotein, anti-DNA, anti-cytoplasmic, antimitochondrial antibodies. There is growing evidence of a role for autoimmunity in graft rejection. Antivimentin antibodies and antibodies to heat shock protein 60 have been associated with chronic rejection in kidney and heart transplants. Our team previously demonstrated that antivimentin antibodies may be associated with CAN.11 Autoantibodies against angiotensin II receptor type 1(AT1) have been implicated in acute humoral rejections in renal transplant patients.12 Opelz et al described panel reactive antibody reactivity to be strongly associated with chronic graft loss in kidney transplants from HLA-identical sibling donors.13 From these findings, we concluded that autoimmunity has a strong role in clinical transplantation. Citrate synthase is a key enzyme in the citric acid cycle, which plays a central role in the regulation of energy homeostasis and cell metabolism; meanwhile it is an important mitochondrial autoantigen.14 There are only sparse Table 1. Values of IgG Isotype CS Autoantibody (⌬OD) in Different Groups at Three Posttransplant Time Points Groups
Autologous (Lewis) Autologous (Fisher344) Vehicle Cyclosporine FK506 Mycophenolate mofetil
4 Weeks
8 Weeks
12 Weeks
0.012 ⫾ 0.008 0.010 ⫾ 0.006 0.013 ⫾ 0.002 0.009 ⫾ 0.006 0.011 ⫾ 0.000 0.015 ⫾ 0.004 0.068 ⫾ 0.007 0.081 ⫾ 0.009 0.083 ⫾ 0.003 0.056 ⫾ 0.006
0.171 ⫾ 0.015 0.137 ⫾ 0.001 0.151 ⫾ 0.011 0.133 ⫾ 0.000
0.335 ⫾ 0.009 0.279 ⫾ 0.019 0.291 ⫾ 0.009 0.099 ⫾ 0.007
reports of autoantibodies against CS. In one study, CS autoantibodies of IgM isotype were mainly observed in healthy controls, while IgG was present at higher levels and four times higher frequency in heart transplant patients.15 This imbalance may result in a shift toward an unfavorable, destructive, autoimmune response that, as the literature suggests, may produce allograft vasculopathy.16 Detection of posttransplantation levels of anti-CS antibodies may help us to follow immune-mediated injury. Autoantibodies of IgM isotype are usually not pathogenic and do not correlate with graft outcome.17 Low-affinity IgM isotype autoantibodies present in human circulation may protect organs from a targeted immune response.18 In our study, IgM isotype CS autoantibody occurred in the sera of all recipients before and after renal transplantation. These antibodies were relatively stable, consistent with “the cognitive theory of the immune system” of discrimination between “physiological” and “pathological” self-reactive antibodies.16 Our study examining the progression of CAN after kidney transplantation revealed the levels of IgG-type CS autoantibody to increase dramatically, a phenomenon that suggests that IgG isotype CS autoantibody may play a role in the development and progression of CAN. Immunosuppresants have different effects on the formation of IgG-type CS autoantibodies; MMF seemed to inhibit the formation of IgG-type CS autoantibodies, whereas CsA and FK506 did not. In conclusion, our study suggested that IgG isotype CS autoantibody contributes to CAN after kidney transplantation, but IgM isotype autoantibody may be physiological. MMF seemed to significantly inhibit the formation of IgG isotype CS autoantibody, which may relate to its effects to alleviate CAN. REFERENCES 1. Pascual M, Theruvath T, Kawai T, et al: renal transplantation. N Engl J Med 346:580, 2002 2. Ojo AO, Hanson JA, Wolfe RA, et al: Kidney Int 57:307, 2000 3. Paul LC: Kidney Int 56:783, 1999 4. Nankivell BJ, Borrows RJ, Fung CL, et al: N Engl J Med 349:2326, 2003 5. Kamada NA: Transplantation 39:93, 1985 6. Lu YP, Xin YP, Gao R, et al: Transplantation 78(suppl):741, 2004 7. Meier-Kriesche HU, Schold JD, Kaplan B: Am J Transplant 4:1289, 2004 8. Rose ML: Curr Opin Organ Transpl 9:16, 2004 9. Cook DJ: Curr Opin Organ Transpl 7:157, 2002 10. Holgersson SS, Holgersson J: Curr Opin Organ Transpl 11:425, 2006 11. Yang L, Lu YP, Luo GH, et al: Transplant Proc 40:2786, 2008 12. Dragun D, Muller DN, Brasen JH, et al: N Engl J Med 352:558, 2005 13. Opelz G: Lancet 365:1522, 2005 14. Nemeth P, Small WC, Evans CT, et al: J Mol Recognit 4:77, 1991 15. Petrohai A, Nagy G, Bosze S: Transpl Int 17:834, 2005 16. Poletaev AB: Biochemistry 67:600, 2002 17. Bran CF, Martinez J, Muruve N, et al: Clin Transpl 15(suppl 6):28, 2001 18. Blass S, Engel JM, Burmester GR: Z Rheumatol 60:I, 2001
From the Transplantation Institute, West China Hospital, Chengdu, Sichuan, China. This study was supported by a grant from the Chinese National Basic Science Foundation (2003CB515504), a grant from the Doctor-Foundation (JYB00402051025), the Program for Changjiang Scholars and Innovative Research Team in
University, Chinese Educational Ministry, and a grant from Shanghai Roche Pharmaceutics Ltd. Address reprint requests to Prof Yi Ping Lu, Transplantation Institute, West China Hospital, Sichuan University, Guoxuexiang 37, Chengdu, China 610041. E-mail: [email protected]
0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.08.071
© 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
4366
Transplantation Proceedings, 41, 4366 – 4368 (2009)
CITRATE SYNTHASE AUTOANTIBODIES
4367
inhibited the formation of IgG isotype CS autoantibody, which may be related to its effects to alleviate CAN. HRONIC ALLOGRAFT NEPHROPATHY (CAN) is one of the major causes of late renal graft loss.1,2 The prevalence of CAN is as high as 60% to 70% on protocol biopsies after the first year.3,4 However, the complex causes of CAN, which involve immunological and nonimmunological factors, have not been fully elucidated. Citrate synthase (CS) is a key enzyme in the citric acid cycle. It plays a central role in the regulation of energy homeostasis and cell metabolism; meanwhile, it is an important mitochondrial autoantigen. In this study, we sought to investigate the role of CS-specific autoantibodies in rat renal allografts displaying CAN.
C
MATERIALS AND METHODS Animals Male inbred Fisher (F344; RT1lv1) and Lewis (LEW; RT11) rats weighing 250 to 300 g were obtained from the Experimental Animal Center of Chinese Academy of Science (Shanghai, China). The animals had free access to water and rat chow under standard conditions.
Kidney Transplantation and Experimental Groups Kidney transplantations were performed from F344 to Lewis (allogeneic), Lewis to Lewis and Fisher 344 to Fisher 344 rats (autologous, each n ⫽ 9) following the procedure of Kamada5 with our modification.6 After allograft recipients were first treated with cyclosporine (CsA: 10 mg/ kg⫺1d⫺1 ⫻ 10 d), they were divided into four groups (each n ⫽ 9): (1)vehicle: vehicle orally; (2)CsA: 6 mg/kg⫺1d⫺1; (3)FK506: 0.15 mg/kg⫺1d⫺1; (4) mycophenolate mofetil (MMF): 20 mg/kg⫺1d⫺1. At 4, 8, and 12 weeks posttransplant, the hosts were sacrificed to harvest sera and renal allografts. The serum creatinine (SCr) was measured and pathological changes assessed according to the Banff 97 criteria.
Assessment of Renal Morphology Formalin-fixed, paraffin-embedded sections of renal tissue samples were stained with periodic acid-Schiff, hematoxylin-eosin, and Masson’s trichrome. All sections were scored based on pathological changes in glomerular, tubular, and vascular tissues upon light microscopy. CAN was determined by two independent nephropathologists according to the Banff 97 criteria.
Detection of Anti-Citrate Synthase Autoantibodies IgM and IgG isotype CS autoantibodies were detected by a binding, indirect, enzyme-linked immunosorbent assay. Citrate synthase from porcine heart (Sigma) was coated (2.5 g/well) overnight at 4 °C, which was followed by a 1-hour incubation at 37 °C on 96-well polystyrol plates (NUNC, Denmark). The plates were blocked using 20% bovine serum albumin in carbonate-buffered saline at 4 °C for 2 hours. Serum samples diluted 1:50 were incubated for 1 hour; before the plates were incubated for 60 minutes with secondary antibodies of rabbit anti-rat IgG horseradish peroxidase (HRPO) or rabbit anti-rat IgM horseradish peroxidase (Sigma). The color reaction using ortho-phenylenediamine
(Sigma) was measured on a Thermo Multiskan MK3 (USA) microplate reader at 450 nm. Results were expressed as OD: namely, the OD from a postoperative sample minus the OD obtained in a reference preoperative sample from the same rat that had been obtained 1 week before the operation.
Statistical Analysis All data shown as mean values ⫾ SD were analyzed by Student’s t tests with comparisons between groups also performed by one-way analysis of variance. P ⬍ .05 was considered significant. We employed SPSS13.0 statistical software.
RESULTS Changes in the Renal Graft Function
Renal allograft function in autologous groups was stable, namely, SCr values (mol/L) of 67.67 ⫾ 6.43, 67.76 ⫾ 4.14, and 70.99 ⫾ 8.19 (Lewis) and 71.23 ⫾ 5.37, 69.13 ⫾ 4.26, and 70.42 ⫾ 7.78 (Fisher344) at 4, 8, and 12 weeks, respectively (P ⬎ .9). At 4 weeks, the SCr in vehicle, CsA, FK506, and MMF groups were 110.97 ⫾ 55.83, 75.27 ⫾ 8.91, 86.07 ⫾ 18.13, and 72.41 ⫾ 7.04, respectively (P ⬎ .05). The SCr obviously increased at 8 weeks and the hosts became uremic at 12 weeks, namely, SCr levels of 275.23 ⫾ 23.51, 295.26 ⫾ 32.07, and 263.64 ⫾ 50.97 at 8; and 516.78 ⫾ 31.59, 525.18 ⫾ 33.67, and 476.93 ⫾ 61.63 at 12 weeks in vehicle, CsA, and FK506 groups, respectively. The SCr values in the MMF group were 138.26 ⫾ 28.48 and 305.94 ⫾ 32.92 at 8 and 12 weeks, respectively, which were significantly lower than those in other three groups (P ⬍ .01). Changes in the Renal Graft Morphology
There were no signs of acute rejection in any group. There was no evidence of CAN in the autologous groups. The Banff scores were 0.33 ⫾ 0.58, 0.67 ⫾ 0.58, and 1.33 ⫾ 0.26 (Lewis) (P ⬎ .05); 0.27 ⫾ 0.58, 0.71 ⫾ 0.45, and 2.33 ⫾ 0.58 (Fisher 344) at 4, 8, and 12 weeks, respectively. At 4 weeks the Banff scores in the vehicle, CsA, FK506, and MMF groups were 1.67 ⫾ 0.58, 1.33 ⫾ 0.58, 1.00 ⫾ 1.00, and 0.67 ⫾ 0.58, respectively. The differences were not significant (P ⬎ .05). The typical pathologic changes of CAN, including vascular and glomerular sclerosis as well as interstitial fibrosis, appeared at 8 weeks in vehicle, CsA, and FK506 groups. At 12 weeks, segmental or global glomerulosclerosis was noted in association with increasing tubular atrophy, interstitial fibrosis, intimal proliferation, and gradual luminal occlusion of the cortical vessels in the vehicle, CsA, and FK506 groups, namely, Banff scores of 5.33 ⫾ 0.58, 12.67 ⫾ 1.16; 4.67 ⫾ 1.53 and 13.33 ⫾ 1.53; 5.67 ⫾ 0.58 and 12.33 ⫾ 3.79 at 8 and 12 weeks, respectively. At 8 and 12 weeks the Banff scores of the MMF group were 1.67 ⫾ 1.16 and 6.67 ⫾ 1.53, respectively, which were significantly lower than those of other three allograft groups (P ⬍ .05).
4368
ZHANG, LU, YANG ET AL
Anticitrate Synthase Autoantibodies
Both IgM and IgG isotype CS autoantibodies were detected in the sera of all recipients before and after transplantation, but the levels of IgM CS autoantibody were obviously higher than those of the IgG isotype. After transplantation they were stable not only in the autologous groups, but also in the allograft groups. At 4 weeks the differences in the values of IgG isotype CS autoantibody (⌬OD) in vehicle, CsA, FK506, and MMF groups were not significant (P ⬎ .05). With progression of CAN at 8 and 12 weeks, the values of IgG (⌬OD) also increased dramatically in vehicle, CsA, and FK506 groups (P ⫽ .005), but the differences were not significant (P ⬎ .05). The levels of IgG (⌬OD) in the MMF group were significantly lower than those in the other three groups (P ⫽ .02; Table 1). DISCUSSION
Despite improvements in immunosuppression, CAN still causes most kidney allograft losses and remains the central clinical challenge.7,8 In spite of the development of sensitive anti-HLA screening assays and crossmatching techniques, there are antibody-mediated rejection episodes as confirmed by C4d deposition, suggesting a role for non-HLA antibodies.9 The non-HLA antibodies associated with solid organ transplantation are a variety of autoantibodies commonly found in autoimmune diseases,10 such as anti-nuclear, anti-nucleoprotein, anti-DNA, anti-cytoplasmic, antimitochondrial antibodies. There is growing evidence of a role for autoimmunity in graft rejection. Antivimentin antibodies and antibodies to heat shock protein 60 have been associated with chronic rejection in kidney and heart transplants. Our team previously demonstrated that antivimentin antibodies may be associated with CAN.11 Autoantibodies against angiotensin II receptor type 1(AT1) have been implicated in acute humoral rejections in renal transplant patients.12 Opelz et al described panel reactive antibody reactivity to be strongly associated with chronic graft loss in kidney transplants from HLA-identical sibling donors.13 From these findings, we concluded that autoimmunity has a strong role in clinical transplantation. Citrate synthase is a key enzyme in the citric acid cycle, which plays a central role in the regulation of energy homeostasis and cell metabolism; meanwhile it is an important mitochondrial autoantigen.14 There are only sparse Table 1. Values of IgG Isotype CS Autoantibody (⌬OD) in Different Groups at Three Posttransplant Time Points Groups
Autologous (Lewis) Autologous (Fisher344) Vehicle Cyclosporine FK506 Mycophenolate mofetil
4 Weeks
8 Weeks
12 Weeks
0.012 ⫾ 0.008 0.010 ⫾ 0.006 0.013 ⫾ 0.002 0.009 ⫾ 0.006 0.011 ⫾ 0.000 0.015 ⫾ 0.004 0.068 ⫾ 0.007 0.081 ⫾ 0.009 0.083 ⫾ 0.003 0.056 ⫾ 0.006
0.171 ⫾ 0.015 0.137 ⫾ 0.001 0.151 ⫾ 0.011 0.133 ⫾ 0.000
0.335 ⫾ 0.009 0.279 ⫾ 0.019 0.291 ⫾ 0.009 0.099 ⫾ 0.007
reports of autoantibodies against CS. In one study, CS autoantibodies of IgM isotype were mainly observed in healthy controls, while IgG was present at higher levels and four times higher frequency in heart transplant patients.15 This imbalance may result in a shift toward an unfavorable, destructive, autoimmune response that, as the literature suggests, may produce allograft vasculopathy.16 Detection of posttransplantation levels of anti-CS antibodies may help us to follow immune-mediated injury. Autoantibodies of IgM isotype are usually not pathogenic and do not correlate with graft outcome.17 Low-affinity IgM isotype autoantibodies present in human circulation may protect organs from a targeted immune response.18 In our study, IgM isotype CS autoantibody occurred in the sera of all recipients before and after renal transplantation. These antibodies were relatively stable, consistent with “the cognitive theory of the immune system” of discrimination between “physiological” and “pathological” self-reactive antibodies.16 Our study examining the progression of CAN after kidney transplantation revealed the levels of IgG-type CS autoantibody to increase dramatically, a phenomenon that suggests that IgG isotype CS autoantibody may play a role in the development and progression of CAN. Immunosuppresants have different effects on the formation of IgG-type CS autoantibodies; MMF seemed to inhibit the formation of IgG-type CS autoantibodies, whereas CsA and FK506 did not. In conclusion, our study suggested that IgG isotype CS autoantibody contributes to CAN after kidney transplantation, but IgM isotype autoantibody may be physiological. MMF seemed to significantly inhibit the formation of IgG isotype CS autoantibody, which may relate to its effects to alleviate CAN. REFERENCES 1. Pascual M, Theruvath T, Kawai T, et al: renal transplantation. N Engl J Med 346:580, 2002 2. Ojo AO, Hanson JA, Wolfe RA, et al: Kidney Int 57:307, 2000 3. Paul LC: Kidney Int 56:783, 1999 4. Nankivell BJ, Borrows RJ, Fung CL, et al: N Engl J Med 349:2326, 2003 5. Kamada NA: Transplantation 39:93, 1985 6. Lu YP, Xin YP, Gao R, et al: Transplantation 78(suppl):741, 2004 7. Meier-Kriesche HU, Schold JD, Kaplan B: Am J Transplant 4:1289, 2004 8. Rose ML: Curr Opin Organ Transpl 9:16, 2004 9. Cook DJ: Curr Opin Organ Transpl 7:157, 2002 10. Holgersson SS, Holgersson J: Curr Opin Organ Transpl 11:425, 2006 11. Yang L, Lu YP, Luo GH, et al: Transplant Proc 40:2786, 2008 12. Dragun D, Muller DN, Brasen JH, et al: N Engl J Med 352:558, 2005 13. Opelz G: Lancet 365:1522, 2005 14. Nemeth P, Small WC, Evans CT, et al: J Mol Recognit 4:77, 1991 15. Petrohai A, Nagy G, Bosze S: Transpl Int 17:834, 2005 16. Poletaev AB: Biochemistry 67:600, 2002 17. Bran CF, Martinez J, Muruve N, et al: Clin Transpl 15(suppl 6):28, 2001 18. Blass S, Engel JM, Burmester GR: Z Rheumatol 60:I, 2001