In kidney transplantation, chronic rejection is the most important cause of late graft loss (1 2,3 4 5
The multifunctional cytokine transforming growth factor-β (TGF-β) is believed to play an important role in allograft rejection. The histologic lesions seen in chronic rejection are the result of excessive deposition of extracellular matrix (ECM) components, a process thought to be mediated to a large extent by the effects of TGF-β (6 7,8 9 10 11–14
Studies in humans and animal models have shown altered mRNA and protein levels of TGF-β and ECM molecules in kidneys with acute or chronic allograft rejection compared with those in control tissue (15–19
METHODS
Patients and Study Design
This study involved patients who received a cadaveric renal transplant at the Leiden University Medical Center between January 1986 and September 1995. From a study of 654 kidney transplants, described in a previous report (20 20
Patients were classified into two groups based on the late graft event. Patients in one group had a deteriorating graft function beyond 6 months after transplantation, and had lost their graft from a biopsy-proven chronic rejection (CR+ group). The biopsies showed specific obliterate vascular changes in arteries, and presence of interstitial fibrosis and tubular atrophy, according to the Banff 1997 classification (21
From the CR+ group and CR− group we selected patients who had experienced at least one interstitial or vascular acute rejection episode within the first 6 months after transplantation. Treatment of acute rejection consisted of methylprednisolone, antithymocyte globulin, and methylprednisolone for first, second, and third acute rejection episodes, respectively. Further details concerning treatment have been described in a previous report (22
As an additional control, native kidneys with normal function and histology, obtained at autopsy, were studied (n=8). Mean age was 45.9±17.7 years.
Histology
Graft histology in the biopsies studied were diagnostically evaluated using sections stained with periodic acid–Schiff and silver-methenamine. Interstitial acute rejection was diagnosed when a widespread interstitial infiltrate was present affecting the tubules (tubulitis). Vascular acute rejection was diagnosed when arteritis was present. Results were expressed according to the Banff 1997 classification (21
In addition to evaluation of the Banff scores, we evaluated the available sections stained with periodic acid–Schiff, and hematoxylin-eosin, for the total amount of cellular infiltrate in the glomeruli, tubulointerstitium, and vessels together, hence regardless of its location in the tissue. The amount of infiltrating cells was scored on a scale from 1 to 4 (1, weak diffuse infiltrate; 2, some foci; 3, considerable number of foci; 4, extensive number of foci).
RNA Extraction
Cryostat sections were cut from each biopsy. The cortex was pinpointed on the basis of light-microscopic localization of glomeruli in the sections, as described in a previous report (23 (12–15) (Boehringer Mannheim), 0.5 mM dNTP, 1× reverse transcriptase buffer, and 4 U of Omniscript reverse transcriptase (Qiagen, Omniscript cDNA kit, Westbury b.v., Leusden, The Netherlands). Reverse transcription was performed for 60 min at 37°C.
Real-Time Polymerase Chain Reaction
mRNA levels of TGF-β1 , collagen α1(I), collagen α1(IV), decorin, and the household gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the biopsies were quantified by real-time polymerase chain reaction (PCR; Prism 7700 Sequence Detector System Perkin-Elmer, Foster City, CA), as described in detail elsewhere (24 1 ); 5′-ACT CTT TTG TGA TGC ACA CCA-3′, 5′-AAG CTG TAA GCG TTT GCG TA-3′, and 5′-AAT GGC GCA CTT CTA AAC TCC TCC AGG CAG G-3′ (collagen α1(IV)); 5′-CCT CAA GGG CTC CAA CGA G-3′, 5′-TCA ATC ACT GTC TTG CCC CA-3′, and 5′-ATG GCT GCA CGA GTC ACA CCG GA-3′ (collagen α1(I)); 5′-ACA TCC GCA TTG CTG ATA CCA-3′, 5′-AGT CCT TTC AGG CTA GCT GCA TC-3′, and 5′-TCA CCA GCA TTC CTC AAG GTC TTC CTC C-3′ (decorin); 5′-TTC CAG GAG CGA GAT CCC T-3′, 5′-CAC CCA TGA CGA ACA TGG G-3′, and 5′-CCC AGC CTT CTC CAT GGT GGT GAA-3′ (GAPDH). Probe sequences for the transcripts were chosen over an exon-intron junction to prevent amplification of genomic DNA. The 5′ ends of the TGF-β and collagen probes were labeled by the reporter dye tetrachloro-6-carboxyfluorescein, and the 5′ ends of the decorin and GAPDH probes by 6-carboxyfluorescein. The 3′ ends of all probes were labeled by the quencher dye 6-carboxy-tetramethyl-rhodamine. The PCR reaction contained 100 nM (GAPDH) or 80 nM (other transcripts) of probe, 300 nM of both primers, and TaqMan Universal PCR Master Mix (Perkin-Elmer) including 300 μM dNTP, 2.5 mM MgCl2 , 0.5 U of AmpErase UNG, and 1.25 U of AmpliTaq Gold DNA polymerase. Reactions were carried out in optical 96-well reaction plates covered with optical caps (Perkin-Elmer). Amplification cycles were performed at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and at 60°C for 1 min. The point (designated as CT value) at which the fluorescence intensity exceeds the standard deviation of the baseline fluorescence is a measure for the amount of cDNA, and thus mRNA, in the sample. mRNA signals for the different molecules in each sample were standardized for the GAPDH mRNA signal. All measurements were performed in duplicate. Differences between duplicates never varied by more than one CT value. As a standard in each PCR run we used a 5-fold dilution range of 2 pg of plasmid containing the appropriate template.
Statistical Analysis
Independent-samples t tests and chi-square tests were used to evaluate differences in continuous and categorized variables, respectively, between the CR+ and CR− groups. Real-time PCR data are presented as the means of duplicate measurements, and differences between groups were tested with independent-samples t test. We used Pearson tests for evaluation of correlations between mRNA measurements of different transcripts. Analyses were performed with SPSS (version 9.0) software. For all tests, P <0.05 was considered statistically significant.
RESULTS
Clinical Characteristics
The time between transplantation and graft loss because of chronic rejection in the CR+ group (35.3±33.6 months) was significantly lower than the mean follow-up time in the CR− group (93.4±25.0 months, P <0.001). The biopsies used in our mRNA study for the CR+ and CR− groups had been taken 62.2±50.8 and 45.8±43.3 days after transplantation, respectively (not significant). For the CR+ group, five biopsies had been taken during the first acute rejection episode, three during the second, and two during the third. For the CR− group, these numbers were nine, eight, and one, respectively (not significant). Table 1 shows further characteristics of the CR+ and CR− groups. Only urine protein concentration at 6 months (P <0.02) and the total number of acute rejection episodes (P <0.02) significantly differed between the two groups.
Table 1: Clinical characteristics
Histology
In the CR+ group, 20, 10, 50, 10, and 10% of the biopsies were borderline, type 1A, type 1B, type 2A, and type 3 acute rejection, respectively. In the CR− group these percentages were 11, 22, 44, 6, and 17%, respectively. The distribution of the different types of acute rejection in the CR+ and CR− groups was not significantly different.
The average score of the total cellular infiltrate in biopsies of the CR+ group (2.7±0.9) was not significantly different from that in the CR− group (2.7±0.7;Table 2 ).
Table 2: Histology and mRNA levels
Measurements of mRNA Transcripts
The relative mRNA measurements for TGF-β, collagen IV, collagen I, and decorin in the CR+ and CR− groups have been summarized in Table 2 . The table also shows the mean TGF-β to decorin mRNA ratios for the patients in each group. Figure 1 shows the relative mRNA measurements for TGF-β in the CR+, CR−, and control groups. The mean TGF-β mRNA levels in the CR+ and CR− groups were significantly higher (P <0.005 and P <0.03, respectively) than those in the nontransplant controls. The mean TGF-β mRNA level in the CR− group was 3.4 times higher than that in the CR+ group (P <0.04). We did not find a difference in TGF-β mRNA levels between patients with interstitial acute rejection and patients with vascular acute rejection (data not shown).
Figure 1: TGF-β1 mRNA levels in the CR+ group, in the CR− group, and in nontransplant controls. For the CR+ and the CR− groups, cortical TGF-β mRNA levels were measured by real-time PCR in biopsies during acute rejection episodes within the first 6 months after transplantation. The CR+ group (n=10) contains patients who showed progressive deterioration of graft function beyond 6 months, and developed chronic rejection. The CR− group (n=18) includes patients who retained stable graft function beyond 6 months for at least 5 years. The nontransplant control group consists of seven kidneys, obtained at autopsy, from patients with normal kidney function. mRNA levels are shown as relative levels to the mean mRNA level in the CR+ group, which has been set to 1. Data are expressed as means of duplicate measurements. *P <0.05, #P <0.005.
Together with a higher mean mRNA level for TGF-β in the CR− group compared with that in the CR+ group, there was a 4.2 times higher mean collagen IV mRNA level (P <0.05;Fig. 2A ), a 5.1 times lower mean collagen I mRNA level (Fig. 2B ), and a 3.2 times lower mean decorin mRNA level (P <0.05) in the CR− group (Fig. 3A ). We did not find a difference in mRNA levels for these ECM components between patients with interstitial acute rejection and patients with vascular acute rejection (data not shown). The mean of the TGF-β to decorin mRNA ratios, calculated for each patient, did not significantly differ between the CR+ and CR− groups (Fig. 3B ). The mean decorin mRNA level in the healthy, nontransplant controls was the highest (Fig. 3A ). Consequently, in these controls the mean TGF-β to decorin mRNA ratio was the lowest (Fig. 3B ).
Figure 2: (A) Collagen α1(IV) mRNA levels and (B) collagen α1(I) mRNA levels in the CR+ group, in the CR− group, and in nontransplant controls. Experimental setup was as outlined in the legend to
Figure 1 . *
P <0.05.
Figure 3: (A) Decorin mRNA levels and (B) TGF-β to decorin mRNA ratios in the CR+ group, in the CR− group, and in nontransplant controls. Experimental setup was as outlined in the legend to
Figure 1 . *
P <0.05.
In the CR+ and CR− groups together, there were significant correlations of TGF-β mRNA with collagen IV mRNA (r =0.82, P <0.001), with collagen I mRNA (r =0.57, P <0.005), and with decorin mRNA (r =0.80, P <0.001). The TGF-β to decorin mRNA ratio did not show significant correlation with any of the mRNA measurements.
Correlations of TGF-β mRNA With Histology
We did not find a correlation between the TGF-β mRNA level and the severity of the acute rejections, as determined by the Banff 1997 classification. In the CR+ and CR− groups together, TGF-β mRNA levels significantly and positively correlated with the total cellular infiltrate score in the biopsies (n=28;r =0.42, P <0.05). We also tested for correlations between TGF-β mRNA and the infiltrate score in biopsies taken from patients who experienced only one acute rejection episode (n=11) versus patients who experienced a second or third acute rejection episode (n=17). There was a significant correlation between TGF-β mRNA and the amount of total cellular infiltrate in patients with a first acute rejection episode (r =0.66, P <0.04), although this correlation was not significant in patients with a second or third acute rejection episode (r =0.31).
DISCUSSION
In kidney transplantation the pathogenesis of chronic rejection, the most common cause of late graft dysfunction, is poorly understood. A considerable number of studies have tried to clarify the mechanism responsible for progression toward chronic failure by analyzing expression levels of TGF-β and ECM components. However, it has not yet been shown whether TGF-β and ECM mRNA levels in the early posttransplantation period are associated with the occurrence of chronic rejection later. In this study we tried to establish whether there is an association between mRNA levels of TGF-β and several ECM molecules during acute rejection episodes within 6 months after transplantation and development of chronic rejection.
We found that the mean mRNA levels of TGF-β, collagen IV, and decorin were high during acute rejection in patients who showed a stable graft function beyond 6 months (CR− group), compared with those in patients of whom graft function deteriorated beyond 6 months and who eventually lost their graft from chronic rejection (CR+ group). It should be noted that when the three highest TGF-β mRNA expressers in the CR− group are left out of our analyses, the mean TGF-β mRNA levels in the CR+ and CR− groups do not significantly differ. This indicates the need for confirmation of our findings in a larger patient group, which will be the goal of a future study. Collagen I mRNA levels did not differ significantly between the CR+ and the CR− groups, probably owing to the large spread of values obtained within the CR− group. The mRNA ratios between TGF-β and decorin, a natural antagonist of TGF-β, did not differ significantly between the CR+ and the CR− groups either. This may indicate that despite the higher TGF-β mRNA levels in the CR− group, TGF-β is not able to exert its effects, because of possible counteraction by decorin. However, we found significant correlations of the levels of mRNA for TGF-β with the levels of mRNA for all ECM components tested during acute rejection, suggesting that the TGF-β message we measured is a reflection of the presence of active TGF-β capable of regulating ECM transcription. Still, as it is not possible to discriminate active from latent TGF-β with the aid of merely quantitative reverse transcriptase PCR, it would be informative to analyze expression levels for molecules such as thrombospondin that have been described as being involved in the activation of TGF-β (25
We considered several explanations that might account for the differences in TGF-β and ECM mRNA levels between the CR+ group and the CR− group: (1) A difference in cyclosporine maintenance therapy between the groups. Cyclosporine has been described to affect TGF-β mRNA expression (26 27,28
The most important observation in this study is that relatively high mRNA levels of TGF-β and ECM components during acute rejection are associated with the absence of chronic rejection. In line with this observation are preliminary data of other authors, who described that a high TGF-β phenotype is associated with a decreased risk of a Banff chronic score of ≥2 in the 6-month biopsy (29 1 into allogeneic cardiac grafts at the time of transplant surgery resulted in prolonged survival of the allografts (13,30,31 1 protein in rats prolonged the survival of cardiac allografts (14 32 11 12 33 34 13 35 36
If our hypothesis that TGF-β production during acute rejection lessens the severity of the episodes is true, one might expect that the rejection episodes in the patients with the highest TGF-β mRNA expression or the highest TGF-β to decorin mRNA ratio are the mildest. Indeed, the three patients with the highest TGF-β mRNA expression have episodes of relatively low severity, i.e., Banff type I. The two patients with the highest TGF-β to decorin ratio also had rejection episodes of Banff type I severity.
Our observations leave open the possibility that TGF-β plays a profibrogenic role during the process of chronic rejection itself, as has been suggested by others in studies of TGF-β gene polymorphism (37,38 17,39
Our results suggest a possible role of TGF-β mRNA as a prognostic marker. More specifically, an increased TGF-β response in early biopsies is correlated with an absence of chronic rejection in the course of time, and might thus indicate a better prognosis. In contrast, a low TGF-β response does not discriminate between those who progress and those who do not progress, given the partial overlap between both study groups (Fig. 1 ). Clearly, the predictive value of TGF-β mRNA levels and the potential beneficial actions of TGF-β in early renal transplant biopsies must be further evaluated in future prospective studies.
In summary, we have shown that mean mRNA levels of TGF-β and ECM, but not histopathologic changes, in biopsies with acute rejection taken within the first 6 months after transplantation differ significantly between individuals who beyond 6 months show deteriorating kidney function and develop chronic rejection and individuals who beyond 6 months show stable kidney function. These mRNA levels are higher during the acute rejection phase in individuals with continuous graft acceptance than those in individuals who develop chronic rejection. Our findings might indicate a beneficial effect of TGF-β during acute rejection in that this cytokine plays an important role in immunosuppression and induction of tissue repair.
Acknowledgments.
The authors thank Paul Eijlers for help with the statistical analyses of the data, and Dr. J.W. de Fijter for critically reading the manuscript.
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