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| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
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| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
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CLINICAL SCIENCE |
*Nephrology Department, Hospital Universitario Bellvitge, L’Hospitalet de Llobregat, Barcelona, Spain; Centre d’Oncologia Molecular, Institut de Recerca Oncológica (COM-IRO), Hospital Duran i Reynals, L’Hospitalet de Llobregat, Barcelona, Spain; Biochemistry Department, Hospital Universitario Bellvitge, L’Hospitalet de Llobregat, Barcelona, Spain; Urology Department, Hospital Universitario Bellvitge, L’Hospitalet de Llobregat, Barcelona, Spain; and ||Transplant Coordination Unit, Hospital Universitario Bellvitge, L’Hospitalet de Llobregat, Barcelona, Spain
Correspondence to Dr. Daniel Serón, Nephrology Department, Hospital Universitario Bellvitge, Feixa Llarga s/n, L’Hospitalet de Llobregat, Barcelona E08907, Spain. Phone: +34–932607602; Fax: +34–932607607; E-mail: 17664dsm{at}comb.es
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Abstract |
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Introduction |
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One of the gene systems that could have a relevant role in the pathogenesis of CAN is the renin angiotensin system (RAS), because its blockade has clearly demonstrated a beneficial effect in different chronic nephropathies (5,6). Consequently, the molecular characterization of gene polymorphisms of the RAS represents an area of intense research to evaluate the susceptibility of the kidney to different diseases and the response to RAS blockade therapies (7), especially regarding the angiotensin I–converting enzyme (ACE), the key regulatory step in the conversion of angiotensin I to angiotensin II. Thus, it is not surprising that a number of polymorphisms of the ACE gene had been associated with the progressive deterioration of the renal function in different kidney diseases (8–10) but not with the increase of their prevalence or incidence (7).
In transplanted kidneys, scant information is currently available on the association of the different ACE genotypes and the appearance of CAN. In this sense, it has been reported that recipient ACE-DD genotype is associated with an increased serum creatinine level at 3 yr, although histologic diagnosis was not available (11). Furthermore, an association between recipient DD genotype and graft survival has been reported in patients with deteriorating renal function, as well as in pediatric recipients, although this association has not been confirmed by others, suggesting that DD genotype could modulate the evolution of renal transplants only in kidneys that already display structural damage (12–15).
Protocol biopsies are currently used to study the natural history of CAN and its relationship to potential risk factors (1,16) and also are useful to design studies aimed to prevent or treat CAN, because they increase the statistical power in comparison with classical epidemiologic studies based on clinical surrogates of long-term graft survival (17,18). Thus, the aim of this study was to analyze whether ACE genotype is associated with the prevalence or incidence of CAN in protocol biopsies or with renal allograft survival. Furthermore, we evaluated the association between renal ACE mRNA levels and ACE genotype.
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Materials and Methods |
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1 yr after transplantation regardless of serum creatinine or proteinuria (17,18). Donor biopsies were obtained between 1995 and 1997 and since 1999. Since 1999, blood samples from kidney allograft donors and recipients were obtained to determine ACE genotype. ACE genotype was also determined in all recipients from which a protocol biopsy was available and came to the outpatient clinic between October and December 2001, regardless of graft function or graft loss. A sample of 72 healthy volunteers and 41 cadaver donors served as controls to study the distribution of ACE genotype in the general population. This study was approved by the Ethics Committee of our hospital, and written informed consent was obtained from all patients.
Study Design
The prevalence of CAN during the first year was estimated considering all patients for whom at least one protocol biopsy was available. In patients with an early and a late protocol biopsy, only the first one was considered for the prevalence analysis.
To estimate the incidence of CAN, we considered only patients with at least two sequential biopsies: (1) a donor and an early protocol biopsy and (2) a donor and late protocol biopsy or (3) an early and a late protocol biopsy. Incidence of CAN was defined as the appearance of CAN in a protocol biopsy in patients with a previous biopsy not displaying CAN.
Definition of Clinical Variables
The following variables were evaluated before transplantation: age and gender of the donor and recipient, number of HLA mismatches, panel-reactive antibodies, and cold ischemia time. After transplantation, we assessed the following variables: immunosuppressive treatment, acute tubular necrosis, acute rejection (defined as an acute rise of serum creatinine that responded to antirejection therapy), serum creatinine, creatinine clearance calculated by means of the Cockroft-Gault formula (19), proteinuria, mean arterial pressure at the time of biopsy, use of antihypertensive drugs and ACE inhibitors, and mean cyclosporine (CsA) or tacrolimus levels at renal biopsy.
Biopsies
We performed wedge preimplantation biopsies and protocol biopsies with a spring-loaded, 16-gauge, automated gun under ultrasound guidance. Two cores of tissue were obtained. One core was processed for conventional histology (1), and the other one was immediately snap-frozen in liquid nitrogen and stored at –80°C for RNA extraction. Renal lesions were scored according to the 1997 Banff working classification criteria (20). All biopsies contained at least one glomerular and one arterial section.
Determination of ACE Polymorphisms
Genomic DNA was extracted from peripheral white blood cells according to standard methods (21). Detection of the deletion (D) and insertion (I) alleles of the ACE gene was performed by amplification of a fragment within intron 16 (22) with 1 µmol of the following primers: (sense 5'-CTGGAGACCACTCCCATCCTTTCT-3' and antisense 5'-GATGTGGCCATCACATTGGTCAGAT-3') in a final volume of 25 µl that contained 50 to 200 ng of genomic DNA. DNA was amplified for 35 cycles at 94°C for 45 s, 60°C for 60 s, and 72°C for 60 s, followed by a final extension at 72°C for 5 min. PCR products were resolved in 1.5% agarose gels and stained with ethidium bromide (band sizes were 192 bp for allele D and 472 bp for allele I). For avoiding mistyping of I/D as a result of insufficient amplification of I allele, an insertion-specific PCR was performed with primers ACE/II-UP 5'-TGGGACCACAGCGCCCGCCACTAC-3' and ACE/II-DOWN 5'-TCGCCAGCCCTCCCATGCCCATAA-3' in the same conditions stated above, except that the extension temperature was 65°C. These primers amplified a 335-bp band.
Real-Time Quantitative PCR Analysis of ACE
Total RNA isolation and reverse transcription were done as described previously (23). Approximately 500 ng of cDNA was amplified (in triplicate) by real-time quantitative PCR (Taq-Man, ABI Prism 7700 Sequence Detection System, Perkin Elmer) using optimized Assays-on-Demand gene expression products (ECA Assay, ID number: Hs00174179_m1; Applied Biosystems). Amplification reactions included 900 nM primers and 250 nM probe (FAM dye-labeled TaqMan-MGB probe) in TaqMan Universal PCR master mix and were performed at universal thermal cycling conditions (a first step of 2 min at 50°C to activate AmpErase-UNG enzyme, a second step of 10 min at 95°C to activate AmpliTaq polymerase, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C) following the manufacturer’s instructions (Applied Biosystems). As internal control for normalization, we amplified the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene with a commercial pair of primers (PDARS; Applied Biosystems). All amplification batches included controls without template and cDNA aliquots from two samples that served as positive controls and were repeatedly quantified to assess interassay variability. ACE mRNA levels were quantified using the delta threshold cycle (CT) method, based in the relative quantification of ACE expression normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase after determining the first cycle of fluorescence detection (threshold cycle) and calculating the differences of these threshold cycles (24,25). Samples with CT values 
36 were excluded from analysis because they were outside the proven linear dynamic range of the assays.
Statistical Analyses
Results were expressed as the mean ± SD. We used the frequency of I/D alleles to perform a Hardy-Weinberg equilibrium test on the patients and the 
2 test to compare the expected and observed genotype frequencies. 2 test was also used to compare qualitative data. T test and ANOVA were used to compare normally distributed quantitative data. Mann-Whitney U test and Kruskal-Wallis test were applied to compare nonparametric data. Patient survival, death-censored graft survival, and death-censored graft survival excluding graft loss for any other reason than CAN were calculated by Kaplan Meier analysis, and the statistical significance of the differences was assessed by the log-rank test. A two-tailed P < 0.05 was considered significant.
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Results |
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The frequency of the D and I alleles of the ACE gene in patients who received a transplant was 66.1% and 33.9%, respectively, with the ACE genotype distribution being in accordance with the Hardy-Weinberg equilibrium.
To study the distribution of ACE genotypes in the normal population, we used 113 control samples, obtained from 72 volunteers without renal disease and from 41 cadaveric renal allograft donors (mean age, 42 ± 13 yr; 49.6% men and 50.4% women). The distribution of ACE genotype in patients who received a transplant was close to being statistically different from the control group (DD = 43.3%, ID = 45.6%, II = 11.1% versus DD = 30.1%, ID = 54.9%, II = 15%, respectively; P = 0.072), although a higher proportion of the ACE-DD genotype was observed in the group that received a transplant (DD = 43.3%, non-DD = 56.7% in patients who received a transplant versus DD = 30.1%, non-DD = 69.9% in controls; P = 0.026).
The baseline characteristics of patients are shown in Table 1. The cause of the ESRD and the immunosuppressive therapy used were not different in the groups defined by the different ACE genotypes.
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10 glomeruli, more than two arteries), 34 (18.9%) biopsies represented minimum sample adequacy (between seven and nine glomeruli and at least one arterial section), and 27 (15%) biopsies were inadequate (between one and six glomeruli and at least one arterial section). Banff diagnosis distribution was not different in the recipients grouped according to ACE genotype (Table 2). When patients were classified according to the presence or absence of CAN, ACE genotype distribution was also not different (data not shown). Furthermore, there was no association between CAN grade and ACE genotype distribution. Banff scores of the biopsies, grouped according to the ACE genotype of recipients, were also not different (Table 3). Finally, no significant differences were observed in the characteristics of patients at the time of biopsy between the various ACE genotype groups (Table 4).
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ACE Genotype and CAN Incidence
To evaluate the association between ACE genotype and CAN incidence, we analyzed 152 patients with at least two sequential biopsies: (1) a donor and an early protocol biopsy (n = 77), (2) a donor and a late protocol biopsy (n = 55), and (3) an early and a late protocol biopsy (n = 20). Early protocol biopsies were performed 153 ± 46 d and late protocol biopsies at 456 ± 93 d after transplantation. Progression to CAN was observed in 42 patients, 83 cases did not progress, and 27 cases showed CAN in both biopsies. We did not observe any significant difference in the ACE genotype between patients with progression to CAN (DD = 38.1%, ID = 47.6%, II = 14.3%), patients without progression to CAN (DD = 44.6%, ID = 48.2%, II = 7.2%), and patients who showed CAN in both biopsies (DD = 44.5%, ID = 40.7%, II = 14.8%). Even when patients were grouped as DD or non-DD, the incidence of CAN was also not different.
The incidence of CAN according to ACE genotype in the 45 patients who received induction therapy (DD = 62.5%, ID = 37.5%, II = 0%) and in the 107 patients who did not receive induction therapy (DD = 32.4%, ID = 50%, II = 17.6%) was not different. Similar results were observed taking into consideration the maintenance immunosuppressive therapy (DD = 28.6%, ID = 53.6%, and II = 17.8% in 104 CsA-treated patients versus DD = 57.1%, ID = 35.7%, II = 7.2% in 44 tacrolimus-treated patients).
ACE Genotypes and Graft Survival
At 12 yr of follow-up, 29 patients lost their graft for the following reasons: CAN (n = 14), acute rejection after protocol biopsy (n = 5), glomerulonephritis associated with hepatitis C virus (n = 4), recurrence of the primary disease (n = 3), nontreatment compliance (n = 2), and chronic pyelonephritis (n = 1).
Patient survival (DD, 95%; ID, 98%; II, 90%; NS), death-censored graft survival (DD, 33%; ID, 64%; II, 70%; NS), and death-censored graft survival excluding patients who lost their graft for reasons other than CAN (DD, 51%; ID, 78%; II, 100%; NS) were not different in patients who were grouped according to ACE genotypes.
Taking into consideration that the presence of CAN in the protocol biopsy is an independent predictor of graft survival, we analyzed the possible influence of ACE genotype to modulate the progression to renal failure. For this purpose, patients were grouped into six categories: II-normal biopsy, ID-normal biopsy, DD-normal biopsy, II-CAN, ID-CAN, and DD-CAN. As shown in Table 5, there were no clinical differences among groups. However, death-censored graft survival excluding patients who lost their graft for any other reason than CAN was significantly lower in DD-CAN patients.
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We did not observe significant differences in the ACE mRNA levels in protocol biopsies according to recipient ACE genotype (DD = –3.91 ± 2.64-fold, n = 23; ID = –4.57 ± 2.70-fold, n = 34; II = –4.84 ± 1.98-fold in ACE copies, n = 7; NS). Similar results were obtained when patients who were treated with ACE inhibitors (n = 6) were excluded from the analysis (data not shown).
Taking into consideration the influence of ACE genotype in graft survival, we reanalyzed whether ACE mRNA levels were different in the six diagnostic categories (II-normal biopsy, ID-normal biopsy, DD-normal biopsy, II-CAN, ID-CAN, and DD-CAN) despite the small sample size in some groups, and we did not observe any statistical difference among groups (Figure 1). To increase the statistical power, we grouped patients as DD and non-DD genotype according to the histologic diagnosis and observed higher ACE mRNA levels in allografts displaying CAN and ACE-DD genotype when compared with allografts displaying CAN and non-DD genotype (DD = –3.36 ± 2.35, n = 9; non-DD = –5.65 ± 1.72 folds in ACE copies, n = 15; P = 0.012).
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Finally, when we analyzed ACE mRNA levels in the protocol biopsies, considering the different ACE genotypes from donors, we did not observe any significant difference (DD = –4.64 ± 2.17-fold, n = 12; ID = –4.40 ± 3.16-fold, n = 22; II = 0.72 ± 0.41-fold in ACE copies, n = 3; NS). There were no differences between groups when patients who were treated with ACE inhibitors were excluded (data not shown).
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Discussion |
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In the present study, we observed a higher proportion of homozygous DD alleles in renal transplant recipients when compared with a control population without known renal disease. This result is not surprising when we take into consideration that the ACE-DD genotype is more prevalent in patients on dialysis (27), and it has been associated with the progression of chronic renal failure in different renal diseases such as diabetic nephropathy, IgA nephropathy, and polycystic kidney disease (8–10). It is important to remark that ACE-DD genotype has not been related to an increased prevalence or incidence of any of the above mentioned renal diseases but to a poorer outcome once renal structural damage has already appeared. These observations suggest that ACE genotype modulates progression to chronic renal failure only in patients who already display chronic lesions, but that does not increase the susceptibility to renal disease. In our study, we failed to observe any association between ACE genotype and the prevalence or incidence of CAN. In previous studies, it has been shown that donor age, acute rejection, CsA toxicity, and delayed graft function were associated with the presence of CAN in protocol biopsies (1,16). In addition, we did not observe any association among the various immunosuppressive treatments used and the prevalence or incidence of CAN in the various ACE genotype groups. Thus, our data indicate that ACE genotype is not a major risk factor for developing CAN.
Various reports have failed to establish any association between ACE genotype and graft survival in the general population of transplant recipients (12,14,15). However, it has been reported that recipient—but not donor—ACE-DD genotype has a negative impact on the long-term graft survival in patients who are at high risk for graft loss, such as patients with creatinine clearance <50 ml/min or proteinuria 0.5 g/d at 12 mo after transplantation (12). A similar observation has been made in pediatric recipients, a population with a higher risk for developing allograft dysfunction as a result of their increased immune alloresponsiveness, high renin activity, and the ability to activate the RAS pathway after injuries (13). These observations suggest that the ACE genotype would act as a genetic modifier of the progression to chronic renal failure once structural damage is already present. However, in these studies, renal histology was not available, and, consequently, it is not possible to rule out that other causes of graft dysfunction such as nephrotoxicity, acute rejection, and recurrent or "de novo" glomerulonephritis were responsible for renal function deterioration in patients who display the ACE-DD genotype. In our study, we observed that the ACE-DD genotype was associated with a reduced graft survival in patients who already display CAN in the protocol biopsy but not in patients without chronic renal lesions.
Finally, we observed that ACE-DD recipients who displayed CAN in the protocol biopsy had higher renal ACE mRNA levels than non-DD patients with CAN. This result is in agreement with the observation that the ACE-DD genotype is associated with increased ACE levels, although in normal allografts, we failed to find this observation, suggesting that there are other factors that modulate ACE gene expression (28–30). On the contrary, we did not observe any association between ACE mRNA levels and ACE genotype in donors. These observations are in agreement with the observation that recipient—but not donor—ACE genotype had been associated with graft survival (12). Moreover, current data suggest that graft-infiltrating mononuclear cells are the major source of ACE activity in renal tissue after renal transplantation (12), and other potential sources of ACE mRNA, such as epithelial or endothelial cells, may also have a recipient origin (31–33). Thus, we may speculate that patients who have the ACE-DD genotype and already display CAN in the protocol biopsy may obtain the maximum benefit from RAS blockade.
In summary, we observed that the ACE-DD genotype in renal transplant recipients is not associated with an increased prevalence or incidence of CAN. However, the DD genotype in patients with CAN diagnosed by means of a protocol biopsy is associated with higher ACE mRNA levels and poorer graft survival.
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Acknowledgments |
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References |
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1 gene. Transplantation 66: 1014–1020, 1998[CrossRef][Medline]
1, IL-10, TNF-
Fas ligand in renal cortex and medulla. Nephrol Dial Transplant 17: 573–579, 2002
Related Article
J. Am. Soc. Nephrol. 2004 15: 1967-1970.
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), and the extreme values (*). The box represents the interquartile range that contains 50% of values. The whiskers are lines that extend from the box to the highest and lowest values, excluding outliers. A line across the box indicates the median. ACE mRNA levels were calculated using the threshold cycle (CT) method normalized for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as the difference in their CT. Values represent the n-fold differences in the number of ACE transcripts.