Renal allograft survival has improved markedly over the past 10 years, in large part as a result of improvements in immunosuppressive therapy. One year graft survival for cadaveric transplants has increased 3-4% per year since 1987 and the half-life of transplanted kidneys has risen from 7.9 to nearly 10 years (1). This progress, however, has been mediated by incremental advances in early graft survival. The rate of late graft loss occurring after the first year has not changed appreciably (2). Thus, refractory acute rejection (AR*) has become relatively uncommon although chronic rejection (CR) has continued to erode away at graft survival at a relatively constant rate.
The etiology of CR remains speculative (2-4). Its correlation with AR episodes, HLA mismatch, and immunosuppressive noncompliance implies that active T cell mediated immunity is, at least in part, responsible. However, once clinically detected, CR is unresponsive to heightened immunosuppression. This suggests that clinically evident renal impairment occurs late in the CR process, or that nonimmune factors become increasingly important with time. Thus, critical evaluation of immune activity in patients before graft deterioration is required to investigate the role of immune events in CR. In addition, if immunity is implicated, some method for the early detection of patients at risk for CR is needed if immune based therapies are planned.
It is clear that T cells can reside within an allograft without causing acute, clinically obvious injury (5, 6). It is also clear that the cells in an allograft during AR are more active with regard to their proliferative, cytotoxic, and secretory abilities than those present in CR or in stable allografts (7, 8). Yet these apparently passive infiltrates remain to be characterized regarding their chronic significance. This characterization becomes increasingly important as interest grows in late immunosuppressive withdrawal and identification of potentially tolerant patients. If, in fact, CR is a T cell-mediated event that progresses subclinically until a critical amount of injury has accumulated, then identification of intragraft T cell activity may segregate individuals who are at high risk for CR from those who will tolerate weaning of their immunosuppression. This study has been designed to correlate intragraft immune activity as determined by histology and cytokine or T cell receptor gene transcription with evidence of early graft injury. The findings presented here demonstrate that immune activity is evident in a majority of stable, well-functioning renal allografts, and that this activity is associated with deteriorating kidney function.
MATERIALS AND METHODS
Patient population. Forty adult patients participating in two ongoing clinical trials requiring protocol biopsies were invited to participate in the study. Patients were required to have stable renal allograft function, defined as freedom from any episode of rejection or clinically significant drug toxicity and no significant increase in serum creatinine for at least 1 year before enrollment. The mean creatinine was 1.6±0.6 mg/dl with a mean glomerular filtration rate (GFR) of 53 ml/min/1.73 m2. Some patients did have one or more successfully treated episodes of acute rejection or drug toxicity more than 1 year before biopsy early in their posttransplant course. The mean number of rejection episodes was 0.5/patient. Thirty-nine patients were taking oral prednisone. Patients on cyclosporine A (Sandimmune formulation) plus mycophenolate mofetil (n=9) and cyclosporine A plus azathioprine (n=13) were included as were patients taking tacrolimus plus azathioprine (n=13), and tacrolimus plus mycophenolate mofetil (n=2). Two patients were on mycophenolate without a calcineurin inhibitor and one patient was off immunosuppression. The mean age of the study population was 46 (22-64). A smaller group of patients (n=6) undergoing biopsy for acute rejection was also analyzed to provide a point of reference to compare the magnitude of the findings in the stable patients.
Clinical evaluation. All patients had serum creatinine and cyclosporine or tacrolimus trough levels measured the morning of their scheduled protocol biopsy. GFR was measured in each patient by the Division of Nuclear Medicine of the University of Wisconsin using a Tc-99 m-DTPA clearance technique. When possible (n=13), a 24-hr urine collection was obtained for quantitative assessment of urinary protein. The Clinical Laboratory Service of the University of Wisconsin performed all laboratory measurements. Creatinine was reassessed 2 years after biopsy in all patients. Fourteen patients were available for a repeat measurement of GFR 2 years after biopsy.
Renal biopsy. Patients were prospectively enrolled in the study after informed consent was obtained and a University of Wisconsin Institutional Review Board approved consent form was signed for two core biopsies to be taken. Biopsies were performed using standard aseptic precautions and local anesthesia under real-time two-dimensional ultrasonic guidance with an 18-gauge needle core device (Biopty-cut, Bard, Covington, GA). No biopsy related complications occurred. Both biopsy cores were coded and examined under a dissecting microscope to insure that each contained renal cortex with glomeruli. Each core was then divided in half. One-half of each biopsy core was submitted for histological analysis and the remaining half was submitted for transcriptional analysis.
Histological evaluation. Biopsy material submitted for histological analysis was processed using standard histopathological techniques and stained with hematoxylin and eosin. Histological indices were evaluated subjectively by a transplant pathologist in a blinded fashion and included fibrosis, tubular atrophy, and cellular infiltrate. Each parameter was then graded from 0 to 3 (none, mild, moderate, or severe) based on the final signed pathology report.
Transcriptional evaluation. Transcriptional analysis was performed by semiquantitative reverse transcriptase-polymerase chain reaction (PCR) as previously described (8, 9). Primers were designed for the amplification of mRNA sequences for cytokine genes previously shown to be important in acute allograft rejection (tumor necrosis factor-α, interferon-γ, interleukin- (IL) 1β, IL-2, IL-4, IL-6, and IL-8) (8, 9). In addition, a primer pair was used to amplify a mRNA sequence for the γ chain of the CD3 subunit of the T cell antigen receptor (8). This was hypothesized to provide insight into the T cell receptor turn-over rate in the allograft (10, 11) and, as such, serve as an indicator of active T cell recognition as opposed to passive T cell presence. Primer pairs for actin and S26 were used as positive controls (8).
Frozen biopsy fragments were homogenized with a PowerGen 35 micro-homogenizer (Fischer Scientific, Fair Lawn, NJ) for 45 sec in a guanidinium thiocyanate-phenol-chloroform lysis buffer (Ultraspec, Biotecx Laboratories, Inc., Houston, TX). The aqueous phase was collected and RNA precipitated with isopropyl alcohol according to the manufacturer's instructions. The resulting pellet was washed with 75% ethanol, resuspended in diethyl pyrocarbonate-treated water and stored at −70°C. RNA yield was determined by spectrophotometry. No more than 3.0 μg of total RNA was then used for Moloney murine leukemia virus reverse transcriptase catalyzed cDNA synthesis as previously described (8). PCR was performed using primers specifically designed to exhibit homogeneous annealing characteristics under the conditions presented by this study (8, 12). Primer sequences have been previously published (8). Cycle numbers were 28 cycles for actin, 37 cycles for IL-2 and IL-4, and 35 cycles for all other primer pairs.
PCR products were then quantitated by high-performance liquid chromatography separation using a Hewlitt Packard 1050 Series Quaternary Pump, Autosampler and VW Detector and a TosoHaas TSK-GEL DEAE-NPR column using a previously published method (9, 13, 14). Each sample consisted of 40 μl of PCR product diluted with 60 μl of 25 mM Tris-HCl at pH 9.0 (total sample volume 100 μl) of which 95 μl were injected. The two solutions used for the mobile phase were: 1 M NaCl, 25 mM Tris-HCl at pH 9.0 (reservoir A) and 25 mM Tris-HCl, pH 9.0 (reservoir B). The gradient program was as follows: 25-44% A in 30 sec, 44-52% A in 5 min, 52-54% A in 2 min 54-60% A in 17 min 60-100% A in 1 min, 100% A for 2 min, 100-25% A in 13 min and then 25% A for 10 min. The total injection-to-injection time was 50 min. The column was operated a 1 ml/min at room temperature. Absorbance was measured at a wavelength of 260 nm. The area under each high-performance liquid chromatography generated curve served as the raw data for analysis.
Study values were standardized as a ratio with the actin signal from each biopsy. Actin is a relatively constant signal in renal biopsies and as such, serves to control for cDNA synthesis efficiency for each biopsy studied (8). The primer homogeneity and a linear relationship between PCR derived signals and translated protein in the transplant setting have been established by prior investigation (8). Each value was thus reported as a transcriptional index consisting of the study parameter divided by the actin amplified from the same biopsy.
Statistical analysis. All data were coded. Separate investigators, prospectively in a blinded fashion, scored each set of indices. All data were submitted in coded form to the statistician (DMH). Variables were then evaluated by the Spearman rank correlation coefficient (rs) analysis. All analyses were done with SAS statistical software (SAS Institute, Inc., Cary, NC).
Evidence of lymphocytic activity and renal injury is present in most stable allografts. Histological evidence of lymphocytic infiltration was detected in 25% of the stable biopsies studied. In addition interstitial fibrosis was seen in 68% and tubular atrophy in 58% of biopsies. Thus, evidence of renal injury was present in two-thirds of individuals who were stable on clinical grounds. Figure 1 shows an example of these findings.
Technical success in obtaining transcriptional data (as defined by actin and S26 transcription) was achieved in 83% of biopsies studied. CD3γ mRNA was detected in 96% of biopsies that met the criteria for technical success and as such, was a far more sensitive indicator of T cell presence than histological assessment of T lymphocyte infiltration. Cytokine transcripts were also detected in low levels in 79% of these stable grafts. These included IL-2 (14%), IL-6 (28%), IL-8 (21%), interferon-γ (33%) and tunor necrosis factor-α (79%). IL-1β was not detected in any specimen from a clinically stable patient. In keeping with previous studies, all AR grafts showed transcription of all genes evaluated and the degree of transcription exceeded that of the stable grafts in each case (data not shown).
Increasing lymphocytic infiltration correlates with early proteinuria and fibrosis. The degree of the histological lymphocytic infiltrate detected in the stable allograft biopsies correlated with the degree of proteinuria as measured by 24-hr urine collection (P=0.034, rs=0.53; Table 1). Increased infiltration was also found to be associated with worsening interstitial fibrosis (P=0.005, rs=0.44). The functional import of this fibrosis (Table 2) was underscored by a significant positive correlation between this finding and the patient's baseline creatinine (P=0.006, rs=0.43) as well as a negative correlation with the GFR measured on the day of the biopsy (P=0.037, rs=−0.35). The intragraft CD3γ signal also correlated with increasing proteinuria (P=0.043, rs=0.51; Fig. 2), suggesting that increased T cell activity was associated with deteriorating graft function. Correlations between CD3γ and graft fibrosis were not established nor were there any associations between CD3γ and creatinine or GFR. Thus, histological evidence of ongoing lymphocyte activity was relatively insensitive, being detected in a low number of patients, but was clearly associated with damage that had progressed to fibrosis and worsened baseline function. CD3γ transcription was comparatively more sensitive and identified patients with micro-proteinuria before histological fibrosis or changes in creatinine.
Of the cytokines studied, IL-6 held the most consistent negative predictive value for histological injury. IL-6 transcripts were detected in 18% of stable allografts studied. This cytokine correlated with lymphocytic infiltrate (P=0.008, rs=0.42), fibrosis (P=0.011, rs=0.41), and tubular atrophy (P=0.05, rs=0.31). Tubular atrophy also correlated with increasing TNF-α (P=0.025, rs=0.36) and IL-8 (P=0.016, rs=0.39) transcripts. Although IL-2 and interferon-γ have traditionally been found only during periods of acute rejection, they were clearly present in low levels in one third of grafts. There was no correlation between histological injury and either of these transcripts.
Patients treated with tacrolimus showed a trend toward less cellular infiltration (P=0.08) but in general, no differences between cyclosporine treated patients and tacrolimus-treated patients reached statistical significance. Similarly, mycophenolate treated patients tended to have a less pronounced infiltrate (P=0.07) and had less IL-8 message detected (P=0.04). Otherwise, no benefit or detriment could be ascribed to a particular immunosuppressive therapy. No associations were evident between drug levels and any of the parameters evaluated.
Given the low number and remote nature of prior rejection episodes in this patient population, acute rejection episodes did not stratify patients in this study in any way. Also, time from transplantation was not related to any parameter studied as would be expected from the relatively close grouping of patients (24-36 months posttransplant).
Evidence of allograft damage at the time of biopsy correlates with worsening renal function over time. All patients were available for repeat measurement of creatinine 2 years after biopsy. Two patients lost their graft to chronic rejection in the follow-up period and one patient died in a motor vehicle accident. Both of the patients lost due to CR had significant lymphocytic infiltration, chronic changes on their original biopsy and proteinuria. For all patients, both fibrosis (P=0.01, rs=0.46) and tubular atrophy (P=0.01, rs=0.47) on the original biopsy were correlated with declining renal function at follow-up. The presence of a lymphocytic infiltrate trended toward a relationship but did not reach statistical significance (P=0.19). This trend was also seen when CD3γ levels were considered. Fourteen patients were available for repeat GFR determination 2 years after biopsy. When GFR was evaluated, rather than serum creatinine, CD3γ levels at the time of original biopsy correlated with the highest change in GFR over time (P=0.045, rs=0.54). Thus, both histological and transcriptional events that were associated with worse function at the time of biopsy correlated with worsening outcome after 2 years of follow-up.
In this study, we have shown that renal transplant patients frequently have significant histological evidence of lymphocytic infiltration and parenchymal injury despite clinically stable allograft function as determined by serum creatinine. They are in keeping with recent data from Lipman et al. (14) in finding significant immune activation in stable allograft recipients. We have correlated these findings with subtle indicators of suboptimal graft function at the time of biopsy, and worsening graft function after 2 years of observation. Furthermore, we have established an association between early graft injury, and gene products fundamentally related to T lymphocyte antigen recognition, namely the TCR, as well as to cytokines known to be involved in acute allograft rejection. As with the histological parameters, these transcriptionally based parameters have correlated with worsening allograft function. These associations, in combination with the known associations between CR and T cell-dependent processes such as AR, suggest that CR has its foundation in ongoing anti-graft immunity. Together, these data suggest that empirical weaning of immunosuppression based solely on stable creatinine may be unwarranted. It remains to be shown if the parameters studied here can identify patients at risk for CR in a way that will allow for successful therapeutic intervention.
Several PCR-based methods have been described for the detection of intragraft transcription (8, 9, 14-19). Investigators have used these techniques to gain an understanding of the biology of AR, and this, in turn has been used to develop transcriptionally based methods for the diagnosis of AR. This study carries these investigations into clinical problems with less obvious causes or endpoints. Certainly the magnitude of transcriptional changes during AR is much greater than in the more insidious process of CR, but these techniques have been refined to the point that rare transcripts can be reproducibly discriminated from background noise. PCR methods certainly have the potential to be overly sensitive, but given the correlates between transcriptional findings and histological and functional damage, this appears not to be problematic in this study. Thus, the use of methods such as the one described in this report appears to be warranted in CR. In fact, the sensitivity appears to be of potential benefit. It is important to underscore the differences in sensitivity to intragraft inflammation between standard histology and reverse transcriptase-PCR, and in particular, to emphasize the functional correlates of these findings. In this study, those allografts with a histologically significant infiltrate had already progressed to fibrosis. The functional import of this fibrosis was apparent in a worsened GFR, a generally elevated baseline creatinine, and proteinuria. It is likely that these grafts have been irreparably injured. In contrast, evidence of T cell activity, in particular CD3γ transcription, was clearly associated with early proteinuria, but was present before histological changes. Even so, this parameter predicted worse graft function at 2 years.
Although most grafts were quiescent with regard to the transcription of the calcineurin-dependent activating cytokines IL-2 and interferon-γ, approximately one-third were not. Although the IL-6 and IL-8 seen in this study are arguably reactionary cytokines induced by damage that has been done, the same cannot be said of cytokines that are so fundamentally associated with early T cell activation. It is thus possible that changes in clinical management may impact the course followed by these allografts the most. Several studies have recently been performed investigating steroid withdrawal in renal allotransplantation (20-22). The percentage of individuals who fail steroid withdrawal are similar to the percentages of individuals with IL-2 and interferon-γ transcription (approximately one-third). It is tempting to speculate that greatly reducing T cell-based immunotherapy in individuals with active T cell transcription would result in progressive inflammatory changes although heightened immunosuppression would favorably impact these individuals. Long-term multi-drug immunosuppression may be required for a significant number of transplant patients. This hypothesis deserves careful prospective scrutiny.
The etiological implications with regard to CR presented by this study support those from large clinical registry reports. Namely, that CR is related to AR more than to any other single event (1). This does not mean that prevention of AR will prevent CR as they may be parallel processes dependent on the same immunological disparity. None the less, heightened consideration of the use of immunosuppression late after transplantation is appropriate. Certainly, the long-term morbidity associated with prolonged immunosuppression is an additional point of concern.
Given the protracted time course of CR and variety of factors impacting on chronic graft function, many issues regarding the sensitivity and timing of evaluation remain complex. Although CR is exacerbated by many factors related to drug toxicity and other nonimmune events, it is probable that immune factors will predominate as our evaluation becomes increasingly sophisticated. Continued study, possibly taking advantage of new, promising techniques for the evaluation of multiple transcripts from tissue derived RNA is justified (23).
Clinically, these data suggest that the current chronic management of patients after renal transplantation suffers from insensitive measurements of graft directed immune activity. It has long been clear that measurement of creatinine alone is insufficient to prospectively detect CR. Although aggressive functional monitoring (e.g., of microproteinuria) may be more sensitive, it still necessitates a response to renal injury rather than a more proactive course of action. Protocol biopsies may prove beneficial in detecting those individuals with overt intragraft inflammation and at least provide some basis for immunosuppressive management. However, this study has provided evidence that evaluation at a more fundamental level may contribute additional relevant predictive information regarding the well being of an allograft.
Acknowledgments. The authors thank Kate Frambs for expert technical assistance. The views expressed in this article are those of the authors and do not reflect the official policy of the Department of the Navy, the Department of Defense nor the United States Government.
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* Abbreviations: AR, acute rejection; CR, chronic rejection; GFR, glomerular filtration rate; IL, interleukin; PCR, polymerase chain reaction; rs, Spearman rank correlation coefficient.