Study of Transforming Growth Factor-b1 Gene, mRNA, and Protein in Japanese Renal Transplant Recipients K. Saigo, N. Akutsu, M. Maruyama, K. Otsuki, M. Hasegawa, H. Aoyama, I. Matsumoto, T. Asano, and T. Kenmochi ABSTRACT Background. Transforming growth factor (TGF)-b1 may contribute to chronic allograft nephropathy and graft loss; however, the exact molecular mechanism remains unclear. Therefore, we assess the relationship between TGF-b1 gene polymorphisms, expression, and development of allograft nephropathy. Methods. We studied 135 renal transplant recipients at our hospital. TGF-b1 gene polymorphisms (codons 10 and 25) were determined from peripheral blood leukocyte DNA. Plasma TGF-b1 mRNA was measured by real-time polymerase chain reaction and TGF-b1 protein levels were assessed by enzyme-linked immunosorbent assay. The relationship between TGF-b1 genotyping, expression, and rejection and results of renal biopsy were evaluated. Results. The genotype frequency of transplant recipients was 49.6%, 30.4%, and 20.0% for C/T, C/C and T/T at codon 10, 100% for G/G at codon 25, respectively. According to the criteria of Banff ‘97 classification, 24 cases were classified as acute rejection and whose genotypes were 16, 3, and 5 cases for C/T, C/C and T/T at codon 10. Plasma mRNA expression was elevated in 14 cases and decreased in 8 cases after acute rejection. We measured 267 specimens of TGF-b1 protein and there was no relation between amount of TGF-b1 protein and mRNA. Conclusion. Our results suggest that the relationship between plasma TGF-b1 expression and the development of allograft nephropathy remains uncertain. Frequency of allograft rejection differ with TGF-b1 codon 10 genotypes and the high-risk genotype was different from the reports of other countries.
D
ESPITE RECENT ADVANCES in immunosuppressive therapy, allograft rejection and chronic nephropathy are the principal causes of allograft dysfunction and graft failure. Transforming growth factor b-1 (TGF-b1) is a 25-kDa homodimeric protein secreted by many cell lines, including plates, T cells, mesangial cells, and monocytes/macrophages. An inflammatory cytokine, TGF-b1, has immunosuppressive properties, which may be beneficial, as well as profibrotic actions, which may contribute to chronic graft loss. TGF-b1 promotes fibrosis and smooth muscle cell migration from the vessel wall into the intima, leading to arteriosclerosis and luminal obliteration. Some had polymorphisms in the TGF-b1 gene; these patients develop chronic rejection more quickly than the rest of the patients. Therefore, we have assessed the relationship between TGF-b1 gene polymorphism, expression, and the development of allograft nephropathy. 0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.11.139 372
PATIENTS AND METHODS The study population consisted of 135 Japanese renal transplant recipients who had undergone living or cadaveric kidney transplantation between June 2004 and March 2009 at our hospital. All patients provided informed consent for study participation. Immunosuppressive therapy consisted of cyclosporine or tacrolimus, mycophenolate mofetil, prednisolone, and basiliximab. TGF-b1 gene polymorphisms (single nucleotide polymorphisms at codons 10 and 25) were determined from peripheral blood leukocytes DNA by direct sequencing of polymerase chain reaction
From the NHO Chiba East Hospital Surgery, Chiba prefecture, Japan. Address reprint requests to Kenichi Saigo, PhD, MD, NHO Chiba East Hospital Surgery, Nitona 673, Chuo-ku, Chiba, Chiba prefecture, Japan. E-mail: [email protected] ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 46, 372e375 (2014)
TGF-b1 GENE, MRNA, AND PROTEIN
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Fig 1. Specimen sampling schedules. Abbreviations: before, before transplantation; ope, engraftment. Study patients underwent renal protocol allograft biopsies at the time of engraftment and at 1 month posttransplantation and episode biopsy when serum creatinine (Cre) level was elevated >20% from baseline.
products. DNA from Japanese healthy volunteer were analyses with the same procedures. Plasma TGF-b1 messenger RNA (mRNA) was measured by real-time polymerase chain reaction and TGF-b1 protein concentration were assessed by enzyme-linked immunosorbent assay and the measurement protocols are presented in Fig 1. The contribution of polymorphisms of the TGF-b1 gene to renal allograft damage was assessed in episode biopsies and their association with TGF-b1 production.
RESULTS Genotype Frequencies of TGF-b1 Polymorphism
We genotyped 135 renal transplant recipientsd83 males (61.5%) and 52 females (38.5%)dand 976 healthy subjects, 595 males (61.0%) and 381 females (39.0%), for codons 10 and 25 polymorphism of the TGF-b1 gene. Genotype frequencies of TGF-b1 polymorphism (codons 10 and 25) between recipients and healthy controls are presented in Fig 2. All the analyzed genotype was G/G at codon 25 in both groups (1 case of a healthy subject could not analyzed because of the deletion). The gene variants of TGF-b1 (codons 10 and 25) were similar distributed between transplant recipients and healthy subjects.
Although we had 24 biopsy-proven rejection cases, the genotype frequencies of rejected group were lower in C/C (12.5%) compared with no rejection cases (34.2%) or healthy controls (30.4%) groups. Expression of the TGF-b1 Gene Products
TGF-b1 mRNA and protein expression in plasma were quantified according to the sampling protocol shown in Fig 1. We quantified 706 specimens of plasma TGF-b1 mRNA by real-time polymerase chain reaction (range from 5.20 106 to 1.30 108, mean 2.61 107 copy/mg RNA). We detected downgrade expression of TGF-b1 mRNA after renal transplantation as compared before transplantation in 73 cases, whereas upregulated expression was detected in 62 cases. TGF-b1 plasma levels were determined in 267 specimens, range from 0.99 to 43.8 ng/mL and the mean level was 9.21 ng/mL. There was no significant correlation between plasma TGF-b1 mRNA and protein (Fig 3A). Serum creatinine levels were increased in patients with allograft rejection; however, the plasma TGF-b1 mRNA levels were increased in same cases but decreased in others (Fig 3B). DISCUSSION
Fig 2. Genotype Frequencies of TGF-b1 Polymorphism of Japanese Healthy Volunteers, 135 Kidney Transplant Recipients and the Distribution at Codon 10 of Kidney Transplant Recipients According to the Presence or Absence of Its Biopsy-Proven Rejection.
TGF-b1 is an inflammatory cytokine involved in the regulation of the immune response that has been related to the onset of allograft rejection and fibrosis [1e3]. Several studies have shown that TGF-b1 gene polymorphism may confer susceptibility to early acute and chronic allograft rejection in heart, lung, kidney, and metabolic syndrome [4,5]. The distribution of genotype at codons 10 and 25 was presented in Figure 2. We had 24 out of 135 cases of allograft rejection (17.8%) that were defined as part of the Banff working classification of renal transplant pathology [6]. TGF-b1 codon 10 genotype frequencies of allograft rejected cases were 12.5%, 66.7%, and 20.8% for C/C, C/T, and T/T, respectively. Although there are several reports in which T/T at codon 10 of TGF-b1 is thought to be associated with allograft rejection and nephropathy [7e9], and Park et al [10] reported that TGF-b1 lower-producer (codon 10 CC and codon 25 GG) genotypes in the renal transplant
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SAIGO, AKUTSU, MARUYAMA ET AL
Fig 3. Expression of the transforming growth factor TGF-b1 gene products. Expression of plasma TGF-b1 mRNA and protein in transplant recipients (A). Expression level of plasma TGF-b1 mRNA absence (1) and presence (2) of allograft rejection (B).
recipients were associated with recurrent acute rejection episodes, whereas Dhaoudi et al [11] reported TGF-b1 high secretion profile may protect the kidney graft. As indicated in our results, the frequencies of T/T were the same between rejected group and others, and the frequencies of C/C at codon 10 was lower in rejected cases than others. We did not find the genetic variation at codon 25 of TGF-b1; that can be considered a cause of genetic landscape variations in this locus that seems to be very different in Caucasian and Japanese or Chinese population [12,13]. There were several reports that TGF-b1 mRNA levels and protein concentration were increased in allograft in nephropathy and rejection [14,15]. We investigated the impact of TGF-b1 mRNA and protein in plasma in transplant recipients (Fig 2) to detect allograft rejection earlier in a manner less invasive than renal biopsy. As indicated in our results, there was no correlation between mRNA and protein (Fig 3A), the time to correct blood samples to analyze may influence the protein levels because TGF-b1 protein was also released gradually from degenerated platelet. Although Pribylova-Hribova et al [16] reported that significantly enhanced TGF-b1 gene expression was detected in allograft tissue during acute rejection compared with controls, both up- and downregulation of TGF-b1 mRNA were detected in plasma of allograft rejected recipients compared with each ones’ baseline plasma mRNA level (Fig 3B). We could not find an overexpression of plasma TGF-b1 mRNA level during allograft rejection; thus, we assume that overexpression during allograft rejection is limited to allograft tissue.
In conclusion, our study showed that elevation of plasma TGF-b1 mRNA levels is not significantly associated with an increased risk of development of allograft rejection and that genotype at codon 10 of the TGF-b1 gene may influence the susceptibility for allograft rejection in renal transplantation.
REFERENCES [1] Border WA, Noble NA. Transforming growth factor b1 in tissue fibrosis. N Engl J Med 1994;331:1286e92. [2] Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor b in human disease. N Engl J Med 2000;342:1350e8. [3] Baboolal K, Jones GA, Janezic A, et al. Molecular and structural consequences of early renal allograft injury. Kidney Int 2002;61:686e96. [4] Guo Z, Binswanger U, Knoflach A. Role of codon 10 and codon 25 polymorphisms on TGF-B1 gene expression and protein synthesis in stable renal allograft recipients. Transplant Proc 2002;34:2904e6. [5] Aihara K, Ikeda Y, Yagi S, et al. Transforming growth factor e b1 as a common target molecule for development of cardiovascular diseases, renal insufficiency and metabolic syndrome. Cardiol Res Pract; 2011:175381. [6] Racusen LC, Solez K, Colvin RB, et al. The Banff 97 working classification of renal allograft pathology. Kidney Int 1999;55:713e23. [7] Inigo P, Lario S, Campistol JM, et al. Role of transforming growth factor beta-1 gene polymorphisms in the development of chronic allograft nephropathy in renal transplant recipients. Nefrologia 2003;23:312e20. [8] Hueso M, Navarro E, Moreso F, et al. relationship between subclinical rejection and genotype, renal messenger RNA, and plasma protein transforming growth factor-b1 levels. Transplantation 2006;81:1463e6.
TGF-b1 GENE, MRNA, AND PROTEIN [9] Jimenez-Sousa MA, Fernandez-Rodriguez A, Heredia M, et al. Genetic polymorphisms located in TGFB1, AGTR1, and VEGFA genes are associated to chronic renal allograft dysfunction. Cytokine 2012;58:321e6. [10] Park JY, Park MH, Park H, et al. TNF-alpha and TGF-beta1 gene polymorphisms and renal allograft rejection in Koreans. Tissue Antigens 2004;64:660e6. [11] Dhaouadi T, Sfar I, Bardi R, et al. Cytokine gene polymorphisms in kidney transplantation. Transplant Proc 2013;45:2152e7. [12] Watanabe Y, Kinoshita H, Yamada T, et al. A catalog of 106 single-nucleotide polymorphisms (SNPs) and 11 other types of variations in genes for transforming growth factor-beta1 (TGFbeta1) and its signaling pathway. J Hum Genet 2002;47:478e83.
375 [13] Ohtsuka T, Yamakage A, Yamazaki S. The polymorphism of transforming growth factor-beta 1 gene in Japanese patients with systemic sclerosis. Br J Dermatol 2002;147:458e63. [14] Shihab FS, Yamamoto T, Nast CC, et al. Transforming growth factor-b and matrix protein expression in acute and chronic rejection of human renal allografts. J Am Soc Nephrol 1995;62: 286e94. [15] Gaciong X, Koziak K, Religa P, et al. Increased expression of growth factors during chronic rejection of human kidney allograft. Transplant Proc 1995;27:928e9. [16] Pribylova-Hribrova P, Kotsch K, Lodererova A, et al. TGF-B1 mRNA upregulation influences chronic renal allograft dysfunction. Kidney Int 2006;69:1872e9.
D
ESPITE RECENT ADVANCES in immunosuppressive therapy, allograft rejection and chronic nephropathy are the principal causes of allograft dysfunction and graft failure. Transforming growth factor b-1 (TGF-b1) is a 25-kDa homodimeric protein secreted by many cell lines, including plates, T cells, mesangial cells, and monocytes/macrophages. An inflammatory cytokine, TGF-b1, has immunosuppressive properties, which may be beneficial, as well as profibrotic actions, which may contribute to chronic graft loss. TGF-b1 promotes fibrosis and smooth muscle cell migration from the vessel wall into the intima, leading to arteriosclerosis and luminal obliteration. Some had polymorphisms in the TGF-b1 gene; these patients develop chronic rejection more quickly than the rest of the patients. Therefore, we have assessed the relationship between TGF-b1 gene polymorphism, expression, and the development of allograft nephropathy. 0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.11.139 372
PATIENTS AND METHODS The study population consisted of 135 Japanese renal transplant recipients who had undergone living or cadaveric kidney transplantation between June 2004 and March 2009 at our hospital. All patients provided informed consent for study participation. Immunosuppressive therapy consisted of cyclosporine or tacrolimus, mycophenolate mofetil, prednisolone, and basiliximab. TGF-b1 gene polymorphisms (single nucleotide polymorphisms at codons 10 and 25) were determined from peripheral blood leukocytes DNA by direct sequencing of polymerase chain reaction
From the NHO Chiba East Hospital Surgery, Chiba prefecture, Japan. Address reprint requests to Kenichi Saigo, PhD, MD, NHO Chiba East Hospital Surgery, Nitona 673, Chuo-ku, Chiba, Chiba prefecture, Japan. E-mail: [email protected] ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 46, 372e375 (2014)
TGF-b1 GENE, MRNA, AND PROTEIN
373
Fig 1. Specimen sampling schedules. Abbreviations: before, before transplantation; ope, engraftment. Study patients underwent renal protocol allograft biopsies at the time of engraftment and at 1 month posttransplantation and episode biopsy when serum creatinine (Cre) level was elevated >20% from baseline.
products. DNA from Japanese healthy volunteer were analyses with the same procedures. Plasma TGF-b1 messenger RNA (mRNA) was measured by real-time polymerase chain reaction and TGF-b1 protein concentration were assessed by enzyme-linked immunosorbent assay and the measurement protocols are presented in Fig 1. The contribution of polymorphisms of the TGF-b1 gene to renal allograft damage was assessed in episode biopsies and their association with TGF-b1 production.
RESULTS Genotype Frequencies of TGF-b1 Polymorphism
We genotyped 135 renal transplant recipientsd83 males (61.5%) and 52 females (38.5%)dand 976 healthy subjects, 595 males (61.0%) and 381 females (39.0%), for codons 10 and 25 polymorphism of the TGF-b1 gene. Genotype frequencies of TGF-b1 polymorphism (codons 10 and 25) between recipients and healthy controls are presented in Fig 2. All the analyzed genotype was G/G at codon 25 in both groups (1 case of a healthy subject could not analyzed because of the deletion). The gene variants of TGF-b1 (codons 10 and 25) were similar distributed between transplant recipients and healthy subjects.
Although we had 24 biopsy-proven rejection cases, the genotype frequencies of rejected group were lower in C/C (12.5%) compared with no rejection cases (34.2%) or healthy controls (30.4%) groups. Expression of the TGF-b1 Gene Products
TGF-b1 mRNA and protein expression in plasma were quantified according to the sampling protocol shown in Fig 1. We quantified 706 specimens of plasma TGF-b1 mRNA by real-time polymerase chain reaction (range from 5.20 106 to 1.30 108, mean 2.61 107 copy/mg RNA). We detected downgrade expression of TGF-b1 mRNA after renal transplantation as compared before transplantation in 73 cases, whereas upregulated expression was detected in 62 cases. TGF-b1 plasma levels were determined in 267 specimens, range from 0.99 to 43.8 ng/mL and the mean level was 9.21 ng/mL. There was no significant correlation between plasma TGF-b1 mRNA and protein (Fig 3A). Serum creatinine levels were increased in patients with allograft rejection; however, the plasma TGF-b1 mRNA levels were increased in same cases but decreased in others (Fig 3B). DISCUSSION
Fig 2. Genotype Frequencies of TGF-b1 Polymorphism of Japanese Healthy Volunteers, 135 Kidney Transplant Recipients and the Distribution at Codon 10 of Kidney Transplant Recipients According to the Presence or Absence of Its Biopsy-Proven Rejection.
TGF-b1 is an inflammatory cytokine involved in the regulation of the immune response that has been related to the onset of allograft rejection and fibrosis [1e3]. Several studies have shown that TGF-b1 gene polymorphism may confer susceptibility to early acute and chronic allograft rejection in heart, lung, kidney, and metabolic syndrome [4,5]. The distribution of genotype at codons 10 and 25 was presented in Figure 2. We had 24 out of 135 cases of allograft rejection (17.8%) that were defined as part of the Banff working classification of renal transplant pathology [6]. TGF-b1 codon 10 genotype frequencies of allograft rejected cases were 12.5%, 66.7%, and 20.8% for C/C, C/T, and T/T, respectively. Although there are several reports in which T/T at codon 10 of TGF-b1 is thought to be associated with allograft rejection and nephropathy [7e9], and Park et al [10] reported that TGF-b1 lower-producer (codon 10 CC and codon 25 GG) genotypes in the renal transplant
374
SAIGO, AKUTSU, MARUYAMA ET AL
Fig 3. Expression of the transforming growth factor TGF-b1 gene products. Expression of plasma TGF-b1 mRNA and protein in transplant recipients (A). Expression level of plasma TGF-b1 mRNA absence (1) and presence (2) of allograft rejection (B).
recipients were associated with recurrent acute rejection episodes, whereas Dhaoudi et al [11] reported TGF-b1 high secretion profile may protect the kidney graft. As indicated in our results, the frequencies of T/T were the same between rejected group and others, and the frequencies of C/C at codon 10 was lower in rejected cases than others. We did not find the genetic variation at codon 25 of TGF-b1; that can be considered a cause of genetic landscape variations in this locus that seems to be very different in Caucasian and Japanese or Chinese population [12,13]. There were several reports that TGF-b1 mRNA levels and protein concentration were increased in allograft in nephropathy and rejection [14,15]. We investigated the impact of TGF-b1 mRNA and protein in plasma in transplant recipients (Fig 2) to detect allograft rejection earlier in a manner less invasive than renal biopsy. As indicated in our results, there was no correlation between mRNA and protein (Fig 3A), the time to correct blood samples to analyze may influence the protein levels because TGF-b1 protein was also released gradually from degenerated platelet. Although Pribylova-Hribova et al [16] reported that significantly enhanced TGF-b1 gene expression was detected in allograft tissue during acute rejection compared with controls, both up- and downregulation of TGF-b1 mRNA were detected in plasma of allograft rejected recipients compared with each ones’ baseline plasma mRNA level (Fig 3B). We could not find an overexpression of plasma TGF-b1 mRNA level during allograft rejection; thus, we assume that overexpression during allograft rejection is limited to allograft tissue.
In conclusion, our study showed that elevation of plasma TGF-b1 mRNA levels is not significantly associated with an increased risk of development of allograft rejection and that genotype at codon 10 of the TGF-b1 gene may influence the susceptibility for allograft rejection in renal transplantation.
REFERENCES [1] Border WA, Noble NA. Transforming growth factor b1 in tissue fibrosis. N Engl J Med 1994;331:1286e92. [2] Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor b in human disease. N Engl J Med 2000;342:1350e8. [3] Baboolal K, Jones GA, Janezic A, et al. Molecular and structural consequences of early renal allograft injury. Kidney Int 2002;61:686e96. [4] Guo Z, Binswanger U, Knoflach A. Role of codon 10 and codon 25 polymorphisms on TGF-B1 gene expression and protein synthesis in stable renal allograft recipients. Transplant Proc 2002;34:2904e6. [5] Aihara K, Ikeda Y, Yagi S, et al. Transforming growth factor e b1 as a common target molecule for development of cardiovascular diseases, renal insufficiency and metabolic syndrome. Cardiol Res Pract; 2011:175381. [6] Racusen LC, Solez K, Colvin RB, et al. The Banff 97 working classification of renal allograft pathology. Kidney Int 1999;55:713e23. [7] Inigo P, Lario S, Campistol JM, et al. Role of transforming growth factor beta-1 gene polymorphisms in the development of chronic allograft nephropathy in renal transplant recipients. Nefrologia 2003;23:312e20. [8] Hueso M, Navarro E, Moreso F, et al. relationship between subclinical rejection and genotype, renal messenger RNA, and plasma protein transforming growth factor-b1 levels. Transplantation 2006;81:1463e6.
TGF-b1 GENE, MRNA, AND PROTEIN [9] Jimenez-Sousa MA, Fernandez-Rodriguez A, Heredia M, et al. Genetic polymorphisms located in TGFB1, AGTR1, and VEGFA genes are associated to chronic renal allograft dysfunction. Cytokine 2012;58:321e6. [10] Park JY, Park MH, Park H, et al. TNF-alpha and TGF-beta1 gene polymorphisms and renal allograft rejection in Koreans. Tissue Antigens 2004;64:660e6. [11] Dhaouadi T, Sfar I, Bardi R, et al. Cytokine gene polymorphisms in kidney transplantation. Transplant Proc 2013;45:2152e7. [12] Watanabe Y, Kinoshita H, Yamada T, et al. A catalog of 106 single-nucleotide polymorphisms (SNPs) and 11 other types of variations in genes for transforming growth factor-beta1 (TGFbeta1) and its signaling pathway. J Hum Genet 2002;47:478e83.
375 [13] Ohtsuka T, Yamakage A, Yamazaki S. The polymorphism of transforming growth factor-beta 1 gene in Japanese patients with systemic sclerosis. Br J Dermatol 2002;147:458e63. [14] Shihab FS, Yamamoto T, Nast CC, et al. Transforming growth factor-b and matrix protein expression in acute and chronic rejection of human renal allografts. J Am Soc Nephrol 1995;62: 286e94. [15] Gaciong X, Koziak K, Religa P, et al. Increased expression of growth factors during chronic rejection of human kidney allograft. Transplant Proc 1995;27:928e9. [16] Pribylova-Hribrova P, Kotsch K, Lodererova A, et al. TGF-B1 mRNA upregulation influences chronic renal allograft dysfunction. Kidney Int 2006;69:1872e9.
