1 Department of Nephrology and Dialysis, 2 Atherosclerosis and Thrombosis Unit, Casa Sollievo della Sofferenza Hospital, IRCCS, San Giovanni Rotondo, 3 Medical Genetics, University of Foggia, Foggia, 4 Department of Nephrology and Dialysis, Foggia, 5 Department of Nephrology and Dialysis, San Severo and 6 Department of Nephrology and Dialysis, Cerignola, Italy
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Abstract |
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Methods. All patients undergoing intermittent dialysis in an Italian district were enrolled as subjects. From the same area, 1307 individuals served as controls. Genomic DNA was obtained and ACE I/D and PAI-1 4G/5G gene polymorphisms were determined. After a baseline evaluation, patients were followed for 28.8±9.8 months. MIs and other causes of death were recorded.
Results. A total of 461 patients (417 on haemodialysis and 44 on peritoneal dialysis) were investigated. At entry, their mean age was 58.2±16.2 years and dialytic age was 82±69 months. Genotype frequencies were not different between controls and uraemics and, in the latter group, between patients with or without cardiovascular diseases at baseline evaluation. During the follow-up, 22 fatal and 16 non-fatal MIs were recorded (mean incidence 1.99 and 1.45%/year, respectively). The adjusted risk of fatal and total MI was related to the presence at entry of a history of MI [hazard ratios (HR) 4.3; 95% confidence interval (CI) 1.512.0 and HR 6.8; 95% CI: 3.314.0, respectively] and to the PAI-1 4/4 genotype (HR 2.8; 95% CI 1.26.9 and HR 2.1; 95% CI 1.14.2, respectively).
Conclusions. In end-stage renal disease, PAI-1 4G/5G gene polymorphism may have a significant role in the occurrence of fatal and non-fatal MI.
Keywords: ACE; dialysis; genetics; mortality; myocardial infarction; PAI-1
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Introduction |
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It has been suggested that in the general population, plasminogen activator inhibitor-1 (PAI-1) and angiotensin converting enzyme (ACE) play a role in the occurrence of coronary heart disease [3]. PAI-1, produced by the vascular endothelium, is the major inhibitor of tissue-type plasminogen activator (t-PA), and plays a critical role in regulating intravascular fibrinolysis [4]. An increased risk for arterial thrombosis has been associated with high plasma levels of coagulation (fibrinogen, FVII) and fibrinolytic pathway factors (t-PA, PAI-1). That low fibrinolytic activity is related to raised plasma levels of PAI-1 has been documented in some subjects who developed MI [3], and it may also be associated with the insulin resistance syndrome [4]. Angiotensin II generated by ACE has a vasoconstrictive effect, promotes proliferation of smooth muscle cells and determines structural cardiac changes that may lead to myocardial ischaemia and congestive heart failure [5]. Moreover, angiotensin II promotes the production of PAI-1 [6].
Common functional genetic polymorphisms, ACE insertion/deletion (I/D) and PAI-1 4G/5G have been shown to modulate gene expressionsubjects with the D allele or the 4G/4G genotype having, respectively, increased ACE activity [7] or plasma PAI-1 concentrations [8].
In a defined area of southern Italy, the District of Foggia, we prospectively investigated the effect of ACE I/D and PAI-1 4G/5G gene polymorphisms on the occurrence of fatal and non-fatal MIs in all patients who were undergoing intermittent dialysis.
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Subjects and methods |
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From January 1995 to December 1996, we interviewed 1397 apparently healthy employees of the Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Southern Italy. All individuals were Caucasian, and all of their parents and grandparents had been born in the same region. A complete clinical history with emphasis on personal and family history for angina pectoris, MI, ischaemic stroke, peripheral arterial disease and vascular risk factors, was solicited from each person by specially trained staff using a previously described questionnaire [9]. Of this cohort of controls, 66 refused to respond, and in 21 DNA was not obtained due to technical problems. Three controls, all men, had documented evidence of coronary heart disease after their enrolment and were excluded from the analysis. Thus, a total of 1307 apparently healthy individuals without a history of any disease, 739 women (56.5%) and 569 men (43.5%), mean age 37.7±9.0 years, served as controls [9]. Among the controls, 23.7% were smokers and mean cholesterol level was 4.87±0.99 mmol/l. There were no differences between smokers and non-smokers for DD or 4G/4G genotypes.
After approval by the local Ethics Committees, the study was carried out according to the Principles of the Declaration of Helsinki, informed consent having been obtained from all subjects.
Assessment of mortality and cardiovascular events
A complete clinical history with emphasis on personal and family history for angina pectoris, MI, ischaemic stroke, peripheral arterial disease and vascular risk factors, was obtained from all patients by specially trained staff using a previously described questionnaire. Hypertension was defined if a subject was being treated with antihypertensive drugs at the time of examination, or as a mean of two measurements of a systolic blood pressure >140 mmHg or a diastolic blood pressure >90 mmHg in the sitting position on at least three different occasions. Blood pressure measurements were made in the early mornings, after adequate rest. Left ventricular hypertrophy was diagnosed by state-of-the-art echocardiography: two-dimensional, ECG guided M-mode and Doppler echocardiographic measurements were performed, in accordance with the recommendations of the American Society of Echocardiography (for haemodialysis patients, on the day after the second dialysis session of the week). The diagnosis of MI was based on an electrocardiogram, angiographic data when available, or clinical and laboratory (CK and CKmb mass) records. Other cardiovascular diseases, such as arrhythmia or valvulopathy, were diagnosed by means of all of the above evaluations. Heart failure was diagnosed by means of echocardiographic findings (ejection fraction <35%). Subjects with either a positive history for diabetes mellitus or a fasting blood glucose level >140 mg/dl, or a 2-h post-load plasma glucose level >200 mg/dl, were considered diabetic.
After the baseline evaluation, patients were followed for 28.8±9.8 months. All MIs or fatal events were reported. Causes of death and fatal and non-fatal MIs were classified by means of codes in clinical records, based on the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM).
Determination of genotypes
Blood samples were collected and DNA was extracted from peripheral blood leukocytes according to standard protocols.
ACE I/D polymorphism. The ACE I/D polymorphism was evaluated as reported previously [10]. Briefly, an insertion-specific primer pair (hace5a, hace5c) and a sense primer flanking the Alu-type sequence were used simultaneously. One hundred micrograms of genomic DNA were amplified in a PTC100 thermal cycler (MJ Research) in a total volume of 20 µl containing 10, 2.5 and 15 pmol of the Alu-flanking sense primer, the insertion-specific primer and the antisense primer, respectively, and 1.5 mmol/l MgCl2, 50 µmol/l of each dNTP and 0.5 U of Taq polymerase (Promega). After a 40-s denaturation at 94°C, we ran 32 cycles of 30 s at 94°C, 45 s at 68°C and 90 s at 72°C, followed by 3 min at 72°C. The products were identified according to standard procedures. The amplification products were resolved in a 2% agarose gel electrophoresis by a 40 mmol/l Trisacetate buffer pH 7.7 containing 1 mmol/l EDTA, stained with 0.5 µg/ml of ethidium bromide, and were visualized by UV light.
PAI-1 4G/5G polymorphism. The PAI-1 4G/5G polymorphism was evaluated as described [11]. Briefly, a mutated oligonucleotide, which inserts a site for the BslI enzyme within the product of amplification, was synthetized. PCR was carried out on 50 µl volume samples in a Perkin Elmer-Cetus thermal cycler. Each sample contained 0.5 µg of genomic DNA, 15 pmol of each primer, 100 mM of dNTP, 10 mM TrisHCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and 1 U thermostable Taq polymerase. The 30 cycles consisted of steps at 95°C for 1 min, at 60°C for 1 min and at 72°C for 2 min. Then, 20 µl vol of the amplification products were digested for 2.5 h at 55°C with 5 U of the BslI restriction enzyme. The fragments were fractionated by 4% agarose gel electrophoresis, and visualized under UV light.
Statistical analysis
All analyses were performed using the Statistical Package for Social Science (SPSS 6.1 for Macintosh). The significance of any difference in means was evaluated by a non-parametric test, whereas the significance of any difference in proportions was tested by 2 statistics. The allele frequencies were estimated by gene counting, and genotypes were scored. Using a
2 test the observed numbers of different ACE I/D or PAI-1 4G/5G genotypes were compared with those expected for a population by the HardyWeinberg equilibrium. The significance of the difference of observed alleles and genotypes between the groups were determined using the
2 test after grouping homozygous and heterozygous carriers of the ACE I allele and homozygous and heterozygous carriers of the PAI-1 5G allele. Odds ratio (OR) and 95% confidence intervals (CI) were calculated. Hazard ratios (HR) and 95% CI were calculated using Cox proportional hazards stepwise regression models, with centre, sex and age, duration of renal replacement therapy, EDTAERA codes and cardiovascular risk factors at entry (MI, heart failure, hypertension, diabetes mellitus, arrhythmia, valvulopathy and left ventricular hypertrophy) included as covariates. Cardiovascular risk factors were also introduced separately into the Cox model. Statistical significance was considered as P<0.05.
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Results |
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Most of the 417 patients were on haemodialysis, whereas a few, 44, were on peritoneal dialysis. All were enrolled in one of the four dialysis units of the District of Foggia (San Giovanni Rotondo, n=152, 33.0%; Foggia, n=142, 30.8%; San Severo, n=100, 21.7%; and Cerignola, n=69, 14.5%).
The mean duration of follow-up was 28.8±9.8 (mean±SD) months. The main characteristics of patients and controls are shown in Table 1. Primary renal diseases, according to EDTAERA Registry codes, are shown in Table 2
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Overall mortality
During follow-up, 106 deaths were recorded (23.0%). The mortality rate was 8.59%/year and mean survival 77.0%. Cardiovascular causes of death were the most important (n=63, 59.4%). Death also resulted from cachexia (n=26; 24.5%), or other miscellaneous causes (n=17; 16.1%).
Overall mortality was related to a history of diabetes mellitus (OR 3.5; 95% CI 2.05.9), arrhythmias (OR 2.5; 95% CI 1.54.4), heart failure (OR 2.6; 95% CI 1.54.3), hypertension (OR 1.8; 95% CI 1.12.8) and MI (OR 2.4; 95% CI 1.24.8). Multivariate event-free survival analysis was affected by a history of cardiac arrhythmias (HR 2.1; 95% CI 1.33.3), diabetes (HR 2.5; 95% CI 1.63.9), age at entry (HR 1.1; 95% CI 1.01.1) and previous MI (HR 2.1; 95% CI 1.13.8).
Myocardial infarction morbidity and mortality
Total cardiogenic mortality was 4.85%/year. As a whole, 79 cardiac events were recorded: 41 cardiac arrests, 22 fatal and 16 non-fatal MIs (mean incidence 3.71; 1.99; 1.45 %/year, respectively).
The occurrence of a fatal MI during follow-up was significantly related to a history of MI, diabetes mellitus or heart failure. Multivariate event-free survival analysis confirmed the association with previous MI (Table 3). When all MIs were taken into account, in a multivariate event-free survival analysis a history of MI was associated with the occurrence of MI (Table 3
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PAI-1 and ACE polymorphisms
There was no significant difference between subjects and controls in the distribution of I/D ACE genotypes or in the allelic frequencies (Table 4).
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The distribution of genotypes observed showed no significant differences when compared with those predicted from the HardyWeinberg equilibrium for either patients or controls.
ACE DD and PAI-1 4G/4G genotype frequencies were similar among uraemics who died and survivors48.1 vs 42.7% and 24.4 vs 23.1%, respectively. When we only considered MI mortality, similar findings were observed for the ACE DD genotype (52.4 vs 43.5%). On the other hand, differences in PAI-1 4G/4G frequencies were not significant (42.9 vs 25.5%; P=0.078). No significant relationship was found between the ACE DD or PAI-1 4G/4G genotypes and other clinical variables. Using a multivariate survival model, overall mortality was not associated with the ACE DD or PAI-1 4G/4G genotypes. On the other hand, the PAI-1 4G/4G genotype showed a strong relationship with fatal MI (HR 2.8; 95% CI 1.26.9) as well as with the occurrence of any MI (HR 2.1; 95% CI 1.14.2).
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Discussion |
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In these patients there is an over-representation of classical risk factors. Moreover, they face additional risks, such as anaemia, hyperparathyroidism and chronic inflammatory processes [12]. Thus, the impact of the genetic background in determining the overall cardiovascular risk can be expected to be low. A pivotal role has been suggested, however, for genes coding for the proteins of the reninangiotensin system, and of the coagulation and fibrinolytic pathways [13].
In this investigation, were enrolled all uraemics undergoing intermittent renal replacement therapy in a southern Italian geographic district; and have shown a clear correlation between MI mortality, as well as morbidity, and the PAI-1 4G/4G genotype. To the best of our knowledge, this is the first study that reports such relationships. An independent role as a major risk factor associated with MI has been ascribed to the 4G/4G genotype [14], and has also been reported in other high-risk clinical conditions, such as the presence and severity of atheromata in patients who underwent coronary angiography for diagnostic purposes [15]. Likewise, PAI-1 activity is strongly associated with such common metabolic abnormalities as obesity and hyperlipidaemia [16]. Although not all investigators support this hypothesis [17], a recent meta-analysis of all the relevant studies showed the PAI-1 4G/4G genotype to have a significant effect on MI [18].
We know that dialysis treatment is characterized by increased circulating levels of PAI-1 and other endothelial cell glycoproteins, which are considered as a subclinical sign of endothelial injury [19], and that this increase is an independent predictor of coronary artery stenosis [20]. Thus, it is conceivable that PAI-1 may contribute to atheromatous disease by promoting a certain degree of hypofibrinolysis. In uraemic patients, the increase of PAI-1 levels may be a consequence of the endothelial cell injury, caused by the dialysis modalities [19], as well as of the effect of 4G/4G genotype, as in the general population [8]. A third cause of increased PAI-1 levels in uraemics may be their insulin resistance combined with the high prevalence of hypertriglyceridaemia among them [16].
A recent study by Ando et al. [21] drew different conclusions than ours. However, the two studies are quite different, and there are many reasons for their disparity: the study of Ando et al. was cross-sectional (ours is a prospective one); it enrolled a smaller population (149 patients and 100 controls vs 461 and 1307, respectively); last but not least, Japanese and Caucasians are ethnically distinct populations with different genetic backgrounds (PAI-1 genotype prevalence in controls: 5G/5G: 11.2 vs 25.2%; 4G/5G: 45.9 vs 47.6%; 4G/4G: 42.9 vs 27.2%).
In the general population, the I/D polymorphism of the ACE gene has been shown as a major risk factor for MI [22]. It is worth noting the evidence for a direct functional link between the reninangiotensin system and the fibrinolytic system [6]. Moreover, these two polymorphisms have been shown to have a synergistic effect in some clinical conditions such as type II diabetic nephropathy [23], diabetes and macroangiopathy [24].
In a previous study we did not find any difference in the genotype distribution of the ACE I/D polymorphism alleles in uraemics and controls and, among uraemics, in patients with or without arterial hypertension, coronary artery disease or left ventricular hypertrophy [9]. In the present study, the ACE I/D polymorphism was not related to overall mortality or the occurrence of fatal and non-fatal MIs. After reaching end-stage renal failure, patients carrying the DD genotype have appeared to have an increased risk for cardiovascular events [25].
In patients on intermittent dialysis, the percentage of subjects carrying the DD genotype was inversely correlated with the time spent on dialysis. This correlation was not found among individuals who received transplants. In keeping with this, in NIDDM patients, who also had end-stage kidneys, a strong association of the DD genotype and coronary artery disease was shown [26]and the DD genotype frequency decreased with the increasing time spent on dialysis [25]. However, our findings, based on a systematic 3-year follow-up of a large dialysis population, as suggested by Yoshida et al. [25], failed to confirm such a hypothesis. We hypothesize that this discrepancy was due to the relatively low prevalence of diabetic patients in our population (9.3%). In fact, the DD-ACE genotype adversely influences specific cardiovascular diseases, but appears to do so in specific geographical areas and in particular subgroups of patients [27].
We are well aware of the limitations of the present study. The observational design does not determine whether the identified association between PAI-1 4/4 and MI represents a causal relationship or an epiphenomenon. Moreover, a survival selection bias is possible, because of the use of a prevalent, rather than inception, cohort and of the high mean time on dialysis of the subject population. Finally, the results of the study might have suffered of information censoring, because patients at high risk of MI may not have been captured well in the study for premature death.
In conclusion, our study suggests an independent role for PAI-1 4G/5G polymorphism as a cardiovascular risk factor in the dialysis setting. These findings emphasize that the genetic background should be taken into account when evaluating cardiovascular risk in the dialysis population. Whether or not an index combining investigation of PAI-1 4G/5G polymorphism and risk factor(s) would be a better marker than either variable examined alone needs to be evaluated in further prospective studies and in other populations.
Conflict of interest statement. None declared.
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Notes |
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References |
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