Relationship between subclinical rejection and genotype, renal messenger RNA, and plasma protein transforming growth factor-beta1 levels.
ABSTRACT Transforming growth factor (TGF)-beta(1) is increased in allograft rejection and its production is associated with single nucleotide polymorphisms (SNPs).
The contribution of SNPs at codons 10 and 25 of the TGF-beta(1) gene to renal allograft damage was assessed in 6-month protocol biopsies and their association with TGF-beta(1) production. TGF-beta(1) genotypes were evaluated by polymerase chain reaction (PCR)/restriction fragment length polymorphism. Intragraft TGF-beta(1) messenger RNA (mRNA) was measured by real-time PCR and TGF-beta(1) plasma levels were assessed by enzyme-linked immunosorbent assay.
Eighty consecutive patients were included. Allele T at codon 10 (risk ratio, 6.7; P = 0.02) and an episode of acute rejection before protocol biopsy (risk ratio, 6.2; P = 0.01) were independent predictors of subclinical rejection (SCR). TGF-beta(1) plasma levels, but not those of TGF-beta(1) mRNA, were increased in patients with SCR (2.59 ng/mL +/- 0.91 [n = 22] vs. 2.05 ng/mL +/- 0.76 [n = 43]; P = 0.01). There was no association between allele T and TGF-beta(1) plasma or intragraft levels.
Allele T at codon 10 of the TGF-beta(1) gene is associated with a higher incidence of SCR.
New England Journal of Medicine 06/2000; 342(18):1350-8. · 53.30 Impact Factor
New England Journal of Medicine 12/1994; 331(19):1286-92. · 53.30 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Chronic allograft nephropathy is an important cause of graft failure. Many donor and recipient factors contribute to its development. Prospective analysis of these factors has been hindered by the lack of sensitive and specific indicators of renal injury. As a consequence protocol biopsies have been increasingly used in the assessment of renal allograft injury. We performed protocol renal allograft biopsies to prospectively examine the role of important determinants and mediators of chronic allograft nephropathy. A total of 51 consecutive cadaveric renal transplant recipients entered a randomized prospective study of tacrolimus (Tac) versus cyclosporine (CsA) microemulsion based immunosuppression. Study patients underwent protocol renal allograft biopsies at the time of engraftment and at 3, 6 and 12 months post-transplantation. Biopsies were analyzed by quantitative polymerase chain reaction (PCR) for mRNA for transforming growth factor-beta (TGF-beta), thrombospondin, and fibronectin. Measurements of renal structural injury were estimated by quantitative assessment of interstitial fibrosis and glomerulosclerosis. Changes in profibrotic growth factors and renal structural injury were related to donor and recipient determinants by stepwise regression analysis. Longitudinal assessment of renal injury demonstrated an early and progressive increase in mRNA for TGF-beta, thrombospondin (TSP) and fibronectin (FBN): TGF-beta baseline, 1.9 +/- 0.2 log copies; TGF-beta 6 months, 2.5 +/- 0.2 log copies, P < 0.05 6 months vs. baseline; TSP baseline, 1.9 +/- 0.2 log copies; TSP 6 months, 2.4 +/- 0.2 log copies, P < 0.05 6 months vs. baseline; FBN baseline, 2.0 +/- 0.2 log copies; FBN 12 months, 2.3 +/- 0.2 log copies, P < 0.05 12 months vs. baseline. This increase in profibrotic growth factors within the allograft was associated with a significant increase in interstitial fibrosis (Vvi) on renal biopsies: Vvi baseline, 13 +/- 1%; Vvi 3 months, 18 +/- 1%; Vvi 6 months, 28 +/- 2%; Vvi 12 months, 34 +/- 2%; P < 0.05 3, 6, and 12 months vs. baseline. Histological analysis demonstrated chronic allograft nephropathy in 4% biopsies at 3 months, 12% at 6 months and in 49% at 12 months. These changes in renal structure were not associated with any change in creatinine clearance (CCr): CCr 3 months, 56 +/- 2 mL/min, CCr 24 months, 56 +/- 2 mL/min; P=NS. Stepwise regression analysis of key donor and recipient determinants of chronic renal injury identified calcineurin inhibitors and acute rejection episodes as important factors involved in the development of chronic renal injury. In particular, the use of cyclosporine compared to tacrolimus was associated with a tenfold increase in TGF-beta mRNA (TGF-beta mRNA at 6 months, CsA vs. Tac, 3 +/- 0.3 vs. 2 +/- 0.3 log copies, P < 0.05), interstitial fibrosis (Vvi at 6 months, CsA vs. Tac, 33 +/- 4% vs. 24 +/- 2%, P < 0.05). Changes in growth factors and renal structure predicted impaired renal function (CCr at 12 months, CsA vs. Tac, 53 +/- 4 mL/min vs. 62 +/- 2 mL/min, P < 0.05). Similarly, acute rejection episodes were associated with an accelerated development of interstitial fibrosis (Vvi at 6 months, acute rejection vs. no rejection, 34 +/- 3% vs. 25 +/- 2%; P < 0.05), but not with changes in TGF-beta, thrombospondin or fibronectin expression. Our results suggest that structural injury develops early in the natural history of the renal allograft and is mediated, in part, by the early up-regulation of profibrotic growth factors. We have determined that calcineurin inhibitors, in particular cyclosporine, and acute rejection episodes are key factors in the development of renal structural injury.Kidney International 02/2002; 61(2):686-96. · 6.61 Impact Factor
Relationship Between Subclinical Rejection and
Genotype, Renal Messenger RNA, and Plasma Protein
Transforming Growth Factor–?1Levels
Miguel Hueso,1,2Estanis Navarro,2Francesc Moreso,1Violeta Beltra ´n-Sastre,2Francesc Ventura,3
Josep M. Grinyo ´,1and Daniel Sero ´n1
Background. Transforming growth factor (TGF)–?1is increased in allograft rejection and its production is associated
with single nucleotide polymorphisms (SNPs).
Methods. The contribution of SNPs at codons 10 and 25 of the TGF-?1gene to renal allograft damage was assessed in
6-month protocol biopsies and their association with TGF-?1production. TGF-?1genotypes were evaluated by poly-
merase chain reaction (PCR)/restriction fragment length polymorphism. Intragraft TGF-?1messenger RNA (mRNA)
was measured by real-time PCR and TGF-?1plasma levels were assessed by enzyme-linked immunosorbent assay.
rejection before protocol biopsy (risk ratio, 6.2; P?0.01) were independent predictors of subclinical rejection (SCR).
TGF-?1plasma levels, but not those of TGF-?1mRNA, were increased in patients with SCR (2.59 ng/mL ? 0.91
[n?22] vs. 2.05 ng/mL ? 0.76 [n?43]; P?0.01). There was no association between allele T and TGF-?1plasma or
Conclusions. Allele T at codon 10 of the TGF-?1gene is associated with a higher incidence of SCR.
Keywords: Protocol biopsies, Renal transplantation, Subclinical rejection, Single nucleotide polymorphisms, Trans-
forming growth factor–?1.
(Transplantation 2006;81: 1463–1466)
been related to the onset of allograft rejection and fibrosis
(1–4). Eight single nucleotide polymorphisms (SNPs) have
in the first exon showing a relationship with allograft rejec-
codon 10, and a G-to-C transversion at position ?915 that
resulted in a change of arginine to proline at codon 25. The
homozygous presence of the C allele at codon 10 and that of
the G allele at codon 25 have been associated with higher
transplant (7, 11, 12). In renal transplantation, it is not clear
ransforming growth factor (TGF)–?1is a cytokine in-
ble condition. Because the presence of subclinical rejection
and chronic allograft nephropathy evaluated by means of
protocol biopsies is associated with poorer graft survival, we
evaluated the association between SNPs of the TGF-?1gene
and the presence of subclinical rejection (SCR) or chronic
allograft nephropathy (CAN) in protocol biopsies.
secutive single renal transplant procedures performed be-
tween 1999 and 2001. Patients recruited fulfilled the follow-
ing criteria: serum creatinine level less than 300 ?mol/L,
proteinuria (excretion ?1 g/d), stable renal function, and
6-month protocol biopsy available. This study was approved
consent was obtained. In our center, the protocol biopsy was
performed at approximately 4 months until 2000 and at 6
Two cores of tissue were obtained: one was processed
for conventional histologic examination and the other was
immediately snap-frozen in liquid nitrogen and stored at
?80°C for RNA extraction. Renal lesions were evaluated ac-
cording to 1997 Banff criteria (13). The presence of border-
line changes or acute rejection prompted a diagnosis of SCR,
and CAN was diagnosed in cases with ci plus ct scores of at
least 2. Biopsy specimens were blindly evaluated by the same
The determination of SNPs at exon 1 of the TGF-?1
blood cells with use of a polymerase chain reaction (PCR)/
restriction fragment length polymorphism method. Each
PCR sample contained 50–200 ng of genomic DNA and 0.2
?M of the sense 5?CACCACACCAGCCCTGTTC and anti-
TV3 001210), M.H. (FIS 2000/0368), E.N. (FIS PI020766, Marato ´
TV3005310), and F.M. (FIS PI040177); and fellowships to M.H. and
V.B.S. from Fundacio ´ Catalana de Transplantament.
1IDIBELL, Department of Nephrology, Hospital Universitario Bellvitge,
L’Hospitalet de Llobregat, Barcelona, Spain.
2IDIBELL, Molecular Oncology Center, Institut de Recerca Oncolo ´gica
(COM-IRO), L’Hospitalet de Llobregat, Barcelona, Spain.
versitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain.
Address correspondence to: Daniel Sero ´n, M.D., IDIBELL, Departament de
Nefrologı ´a,Hospital Universitario
L’Hospitalet de Llobregat, Barcelona E08907, Spain.
Received 31 August 2005.
Accepted 19 December 2005.
Copyright © 2006 by Lippincott Williams & Wilkins
Transplantation • Volume 81, Number 10, May 27, 2006
amplified for 35 cycles at 94°C for 45 seconds, 58°C for 45
mine the allelic variants of the TGF-?1gene, 10 ?L of the
amplified DNA were digested with 10 U of MspA1I (codon
ethidium bromide staining.
Isolation of total RNA and reverse transcription were
done as previously described (14). Steady-state levels of
TGF-?1were determined in triplicate by real-time PCR (ABI
Prism 7700; Applied Biosystems; Bedford, MA) on 0.5 ?g of
complementary DNA with use of the following primers: for-
900 nM, and the TaqMan probe5=FAM-TGGGTTTCCAC-
CATTAGCACGCG-TAMRA at 200 nM. As internal control
cial pair of primers (PDARS ID number, 4310884E; Applied
ger RNA (mRNA), we constructed a standard curve with a
cloned fragment of the TGF-?1transcript that included the
sequence amplified. In subsequent quantifications, this plas-
mid was used to create an absolute standard curve that was
TGF-?1mRNA was expressed as log number of copies.
Fasting plasma samples were obtained at the time of
protocol biopsy and total TGF-?1protein levels were mea-
bent assay (Quantikine; R&D Systems, Abingdon, UK). The
intraassay coefficient of variation of duplicated samples was
Results were expressed as means ? SD. Differences be-
tween categoric data were evaluated with a 2?2 contingency
ysis of variance were employed to compare normally distrib-
uted quantitative data. The Mann-Whitney U test and
Kruskal-Wallis test were applied for nonparametric data.
Univariate and multivariate logistic regression were em-
ity of histologic diagnoses. P values were corrected for the
number of variables compared according to the Bonferroni
We analyzed protocol biopsies from 80 recipients of a
cadaveric graft. Recipient age was 47 years ? 13, 52 of the 80
patients were men, and there were 11 repeat transplantation
cases. Thirty-three patients received cyclosporine and pred-
nisone with antilymphocytic antibodies (n?7), azathioprine
(n?1), mycophenolate mofetil (n?17), or sirolimus (n?4).
The other 47 patients received tacrolimus and prednisone
with mycophenolate mofetil (n?44) or sirolimus (n?3).
TGF-?1SNP distribution in 72 cadaveric donors served as
controls (age 44 y ? 16; 62% male patients).
Protocol biopsies were performed at 168 days ? 58.
Histologic diagnoses were normal (n?28), borderline
changes (n?8), acute rejection (n?5), CAN (n?24), CAN
with borderline changes (n?12), and CAN with acute rejec-
tion (n?3). The CAN was mild in 29 cases, moderate in nine
cases, and severe in one case. Patients with borderline
changes (n?20) or acute rejection (n?8) were categorized
as having subclinical rejection (SCR; 35%). There were 39
statistics were 0.83 for the diagnosis of SCR (95% CI, 0.69–
0.96; P?0.001) and 0.67 for CAN (95% CI, 0.51–0.84;
Characteristics of patients according to TGF-?1geno-
types are shown in Table 1. The distribution of SNP at codon
Baseline characteristics of patients according to TGF-?1genotypes at codons 10 and 25a
TT (n?26)CT (n?33)CC (n?18)GG (n?68)CG (n?9)CC (n?3)
Panel reactive antibodies >20% (no/yes)
Cold ischemia time (h)
aCorrected P Values were not significant for all comparisons.
Transplantation • Volume 81, Number 10, May 27, 2006
10 was not different between transplant recipients and con-
the distribution of SNPs at codon 25 (CC, n?3; CG, n?9;
GG, n?68 vs. CC, n?2; CG, n?9; GG, n?47). The distribu-
tion of genotypes of the TGF-?1gene followed Hardy-Wein-
The prevalence of SCR was different in transplant re-
46%; CC, 8% in grafts with SCR vs. TT, 28%; CT, 41%; CC,
4%; CG, 14%; GG, 82% in SCR vs. CC, 4%; CG, 10%; GG,
86% in grafts without SCR). Conversely, the prevalence of
17%in grafts without CAN) or codon 25 (CC, 3%; CG, 13%;
GG, 84% in CAN vs. CC, 5%; CG, 10%; GG, 85% in grafts
To further evaluate the association between SCR and
Multivariate logistic regression analysis showed that allele T
before the protocol biopsy (risk ratio, 6.2; 95% CI, 1.5–25.0;
P?0.01) were independent predictors of SCR, adjusting for
the type of anticalcineurinic treatment.
TGF-?1plasma levels were determined in 65 patients
61 patients. TGF-?1plasma levels were increased in patients
with SCR (2.59 ng/mL ? 0.91 [n?22] vs. 2.05 ng/mL ? 0.76
pared with tacrolimus (2.58 ng/mL ? 0.98 [n?20] vs. 2.08
ng/mL ? 0.74 [n?45]; P?0.02). Two-way analysis of vari-
ance showed that only SCR was independently associated
with versus without SCR (2.58 log copies ? 0.76 [n?23] vs.
with cyclosporine or tacrolimus (2.42 log copies ? 0.67
[n?23] vs. 2.41 log copies ? 0.73 [n?38]). Allele T at codon
? 0.83 [n?50] vs. 2.45 ng/mL ? 0.87 [n?13]) or mRNA
TGF-?1levels (2.41 log copies ? 0.67 [n?49] vs. 2.50 log
copies ? 0.85 [n?11]).
ciation between recipient allele T at codon 10 and SCR at 6
that this allele is also more prevalent in other inflammatory
diseases and in liver allograft rejection (10, 15–17). In renal
transplant recipients, an association between donor or recip-
ient SNP at codon 10 and an increased incidence of clinical
acute rejection has been reported (8, 9). These data suggest
though the relative contribution of the donor or recipient is
not well established at present. To the contrary, in a large
epidemiologic study (18), there was no association between
SNPs at codons 10 or 25 and 3-year renal allograft survival.
Because the detrimental effect of SCR on graft failure can be
demonstrated in only patients who undergo long-term fol-
low-up, it cannot be discarded that SNPs at codon 10 may
have an influence on long-term graft outcome (19).
is associated with TGF-?1production. However, TT geno-
type has been associated with higher or with lower TGF-?1
mRNA and protein levels (7, 11, 12, 20). In renal allograft
biopsy specimens obtained in pediatric patients with chronic
allograft dysfunction, Melk et al. (20) failed to observe any
association between TGF-?1SNPs and their intrarenal ex-
pression. In our study, performed in stable grafts, we also
failed to observe this association. However, we observed that
the presence of SCR and immunosuppressive treatment was
associated with circulating TGF-?1protein levels, suggesting
in renal transplantation. In addition, we cannot discard that
the association between allele T and SCR may be caused by
other genes in linkage disequilibrium with TGF-?1.
In summary, we suggest that TGF-?1genotype may
influence the susceptibility for SCR in renal transplantation.
The authors thank Dr. Jordi Bover for discussions on the
topic of this work.
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