Angiotensin-converting enzyme activity and the ACE Alu polymorphism in autosomal dominant polycystic kidney disease

Tina Schiavello1, Valerie Burke2, Nadia Bogdanova3, Piotr Jasik4, Steve Melsom5, Neil Boudville6, Ken Robertson7, Dora Angelicheva1, Bernd Dworniczak3, Marta Lemmens3, Juergen Horst3, Vassil Todorov8, Dimitar Dimitrakov9, Wladyslaw Sulowicz4, Andrzej Krasniak4, Tomasz Stompor4, Lawrence Beilin2, Joachim Hallmayer10, Luba Kalaydjieva1,11 and Mark Thomas6,

1 Centre for Human Genetics, Edith Cowan University, Joondalup, Perth, WA, 2 University Department of Medicine, University of Western Australia, Medical Research Foundation Building, Perth, WA, 3 Institut für Humangenetik, WW-U, Münster, Germany, 4 Department of Nephrology, Jagiellonian University, Cracow, Poland, 5 Department of Radiology, 6 Department of Nephrology, 7 Department of Biochemistry, Royal Perth Hospital, Perth, WA, 8 Clinic of Nephrology and Hemodialysis, University Hospital, Pleven, Bulgaria, 9 Clinic of Nephrology and Hemodialysis, University Hospital, Plovdiv, Bulgaria, 10 Centre for Clinical Research in Neuropsychiatry, Graylands Hospital, Perth, WA and 11 Western Australian Institute for Medical Research, QEII Medical Centre, Nedlands, Perth, WA, Australia



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Previous studies concerning Alu I/D polymorphism in the ACE gene and ADPKD severity have used the Alu genotypes as a representative of the true biological variable, namely ACE activity. However, wide individual and ethnic differences in the proportion of variance in ACE activity explained by the I/D genotype may have confounded these studies. This investigation examines the association between ADPKD severity and ACE in terms of plasma enzyme activity and I/D genotypes in individuals from three different countries.

Methods. Blood samples were collected from 307 ADPKD patients (116 Australian, 124 Bulgarian and 67 Polish) for determination of ACE activity levels and I/D genotypes. Chronic renal failure (CRF) was present in 117 patients and end-stage renal failure (ESRF) in 68 patients.

Results. ACE activity was related to the I/D genotype, showing a dosage effect of the D allele (P=0.006). The proportion of variance due to the Alu polymorphism was 14%. No difference in ACE activity and I/D genotype distribution was found between patients with CRF versus normal renal function (P=0.494; P=0.576) or between those with ESRF versus those without ESRF (P=0.872; P=0.825). No effect of the I/D genotype on age at development and progression to renal failure (CRF; ESRF) was detected in the overall group, and in subgroups based on ethnic origin, linkage status and sex.

Conclusion. ACE is not likely to play a role as a determinant of ADPKD phenotype severity.

Keywords: autosomal dominant polycystic kidney disease; angiotensin converting enzyme; chronic renal failure; end-stage renal failure



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by extensive inter- and intra-familial variation in clinical severity [1]. Since mutations in the PKD1 and PKD2 genes are the primary genetic cause of ADPKD, locus and mutation heterogeneity [2,3], as well as the random nature of the somatic mutations proposed by the "two-hit" model [4] are major contributors to phenotype diversity. Nonetheless, the complex pathogenesis of the disorder suggests that additional modifying factors may play a role. Activation of the renin-angiotensin system (RAS), present already in the early stages of ADPKD pathogenesis [5], could promote renal impairment and cyst growth through intrarenal vascular disease, as well as through the ability of angiotensin II to potentiate growth in tubular epithelial cells [6]. A number of studies have therefore focused on the role of the angiotensin converting enzyme (ACE) and tested the association between different measures of ADPKD severity and an intragenic Alu insertion (I) or deletion (D) polymorphism in the ACE gene. The results have been controversial, with some studies suggesting a greatly increased risk of early kidney failure in individuals homozygous for the D allele [7,8], and others failing to find an effect [9,10]. The basic assumption of such studies is that the Alu genotypes are representative of the true biological variable, namely ACE activity. However, estimates of the proportion of the variance in enzyme activity explained by the Alu polymorphism vary substantially [11,12], and individual and population differences in the genetic control over ACE expression can be expected to have a confounding effect on studies where the I/D genotype is used as a substitute for ACE activity.

In an attempt to address this problem, we have studied subjects originating from three different populations, and assessed the association between ACE and ADPKD severity in terms of both plasma enzyme activity and I/D genotypes.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
The study included 307 ADPKD patients, 154 males and 153 females, aged from 9 to 85 years (mean 45.2±0.9 years). According to ethnic origin, they were grouped into 116 Australian (mostly of British descent), 124 Bulgarian, and 67 Polish. Informed consent was obtained from all participating individuals. The study complied with the ethical guidelines of the institutions involved.

The diagnosis of ADPKD was based on accepted ultrasonographic criteria [13]. Renal function and rate of deterioration were evaluated by serum creatinine (SCr) values, collected retrospectively over periods ranging from 1 to 7.2 years (mean 6.8±0.31). The number of measurements per patient varied between 1 and 16 (mean 3.4±0.15). Deterioration of kidney function was assessed using two end points: chronic renal failure (CRF), defined as SCr level of 150 µmol/l (2.25 mg/dl), and end-stage renal failure (ESRF). In our patient population, 117 individuals had reached CRF and 68 had ESRF.

The definition of hypertension followed WHO criteria [14]. The overall number of hypertensive individuals was 192. Information on the use of ACE inhibitors at the time of the study was available for both the Australian and Polish patients, where 54 out of 85 Australian and all 41 Polish hypertensive subjects received such treatment.

Based on genetic linkage data, the patients were subdivided into 184 PKD1, 38 PKD2 and 85 subjects where linkage analysis was not feasible. Since the probability of linkage to the PKD1 gene is around 90% in affected individuals of European descent [15], the 85 undefined individuals were included in the PKD1 group, adding to a total of 269 subjects. Statistical analysis was performed separately on the definite and on the expanded PKD1 group.

The information on the affected individuals is summarized in Table 1Go.


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Table 1. Characteristics of 307 ADPKD patients

 

Methods
Laboratory analyses
Plasma ACE activity was measured in 92 Australian patients using the kinetic method [16], with a reference range of 23–100 U/l (covariance 3.2% at 125 U/l and 8.1% at 31 U/l).

The ACE I/D polymorphism was detected by PCR amplification as described [17]. To avoid mistyping, all D/D homozygous samples were re-typed using an insertion-specific PCR primer [18]. PCR products were separated on 3% agarose gels and visualized by ethidium bromide staining.

Statistical analysis
ACE genotype frequencies were compared between countries using the Chi-square test. Hardy–Weinberg equilibrium (HWE) was assessed with the probability test in the overall sample and with Fisher's exact test within each of the three populations. The relationship between ACE I/D genotypes and plasma ACE activity was assessed by general linear models (GLM). Analysis of covariance (ANCOVA) was used to correct for treatment with ACE inhibitors and analysis of variance (ANOVA) was used to compare the mean values of plasma ACE activity between individuals reaching CRF before or after age 40 years.

Decline in renal function was assessed by linear regression analysis using the least-squares method, with fitting individual regression lines of time vs 1/creatinine values and extrapolating age at CRF. Kaplan–Meier survival curves were used to calculate cumulative survival to CRF and ESRF. Individuals were grouped according to ACE genotype and compared by means of a two-sided log-rank test.

Differences were considered statistically significant at {alpha} of 0.05 (P<0.05). All analyses were performed using SPSS 9.0 (SPSS Inc, Chicago III).



   Results
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
The I/D polymorphism and plasma ACE activity
In the overall group, the allele and genotype frequencies of the I/D polymorphism (Table 2Go) fell within the range reported previously in Caucasian populations [19,10]. A difference between the three populations was observed, with a lower frequency of the D allele in the Polish sample (P=0.02). No deviation from HWE was seen in the overall sample, nor in any individual population.


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Table 2. I/D genotypes observed in the three populations

 
Plasma ACE activity in all 92 Australian ADPKD patients fell within the reference interval (23–100 U/l). There was a significant correlation between Alu genotypes and enzyme activity, with a mean value of 41.2±5.2 U/l observed in I/I, 59.0±4.6 U/l in I/D, and 71.2±5.8 U/l in D/D subjects (P=0.006). Similar results were obtained after adjustment for the use of ACE inhibitors in the control of hypertension (43.5±6.5 U/l for I/I, 56.2±4.1 U/l for I/D and 69.7±4.5 U/l for D/D; P=0.004). The fraction of the variance of plasma ACE activity explained by the ACE I/D polymorphism was estimated at 14%.

Plasma ACE activity and renal function
The relationship between plasma ACE activity and the development of renal failure was tested in the 92 Australian patients (CRF=37; ESRF=27). The mean ACE activity levels were 61.7±3.8 U/l in the group with normal renal function; 59.8±6.4 in the CRF group; and 57.9±5.5 in subjects with ESRF. The difference was not significant (P=0.494 for CRF vs normal renal function and P=0.872 for ESRF vs lack of ESRF). Similar results were obtained after adjustment for treatment with ACE inhibitors.

The mean plasma ACE activity was 62.6±4.1 U/l in the group of patients reaching CRF before age 40 years (n=45) and 56.3±5.5 U/l among those reaching CRF after age 40 years. The difference was not significant (P=0.352).

The I/D genotype and ADPKD phenotype severity
Risk of renal failure
The distribution of I/D genotypes among the 307 ADPKD subjects was examined by comparing subjects with normal renal function to those with CRF and with ESRF (Table 3Go). No significant differences were found between the three groups: P=0.576 in the comparison of CRF vs normal renal function and P=0.825 for ESRF vs lack of ESRF.


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Table 3. I/D genotypes among ADPKD patients with normal renal function, CRF and ESRF

 

Age at development of renal failure
The relationship between the I/D genotype and development of renal failure was examined in a total of 117 subjects with CRF and 68 with ESRF. No significant differences in the mean age to either CRF and ESRF was found. The mean age at CRF was 40.7±1.9 years for I/I, 39.7±1.1 for I/D and 39.1±1.5 for D/D subjects, P=0.810. The mean age at ESRF was 51.5± 3.0 years for I/I individuals, 55.5±1.6 for I/D and 54.5±1.7 for D/D, P=0.415. This analysis was repeated after grouping the patients according to ethnic origin. Again, no statistically significant differences were found, with P=0.739 and P=0.239 for the Australian patients, P=0.504 and P=0.087 for the Bulgarian and P=0.313 and P=0.184 for the Polish.

Lack of significant effect was observed when the different I/D genotypes were compared, within the entire group of patients using Kaplan–Meier cumulative survival curves to CRF (P=0.815) and to ESRF (P=0.478) (Figure 1Go). Similar results were obtained when Kaplan–Meier survival curves were examined separately for PKD1 and PKD2. Analysis of survival to CRF gave P values of 0.606 in the PKD1 and 0.529 in the PKD2 group. For ESRF, the P values were 0.454 for PKD1 and 0.163 for PKD2. No significant difference was revealed between the definite and expanded PKD1 groups. The separate analysis of male and female individuals with ADPKD also failed to detect significant effects of the I/D genotype on survival to CRF or ESRF.



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Fig. 1. Analysis of the effect of the I/D polymorphism on survival to CRF and ESRF in the overall group of patients. Kaplan–Meier cumulative survival curves showed no significant differences between I/I, I/D and D/D genotypes as regards progression to CRF (P=0.810) and ESRF (P=0.415).

 

Hypertension
The distribution of I/D genotypes was compared between 192 hypertensive and 97 normotensive ADPKD subjects. The I/D distribution for the hypertensive group were: I/I (18%), I/D (51%), D/D (31%), and I/I (12.4%), I/D (57.7%) and D/D (29.9%) for the normotensive group. The analysis failed to reveal a significant difference between the two groups (P=0.384).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The effect of the renin–angiotensin system and specifically of ACE activity on renal disease is an issue of practical significance, as it relates to the therapeutic potential of ACE inhibitors in the control of deterioration of kidney function. The findings reported to date on ADPKD, as well as other kidney disorders, have been controversial.

We have examined the association between the alleles of the Alu polymorphism in the ACE gene and decline in kidney function in 307 ADPKD subjects from three different populations. Inter-population differences did exist in terms of both the frequency of I/D alleles and the ratio of PKD1 to PKD2 patients. However, none of the three populations displayed a significant association between the I/D genotype and ADPKD phenotype severity. Lack of association was also observed for the entire study group. No effect was detected on either the overall risk of renal failure, the age at development of renal failure or the rate of progression from normal function to CRF and to ESRF. The findings were also negative in the separate analysis of patients grouped according to linkage status or sex.

In discussing the results of such association studies, one has to keep in mind their oversimplified design, where a DNA polymorphism in one gene is used to represent the overall effect of an entire pathway which is likely to be under complex control. The approach is simplistic even with the regard to ACE itself. The Alu polymorphism in the ACE gene is considered to be a neutral marker in disequilibrium with another, biologically relevant polymorphism directly affecting ACE activity levels. Strong evidence coming from recent studies suggests that the ACE-linked quantitative trait loci (QTL) are probably located in the 3' region of the gene, where a complexity of intragenic haplotypes can be observed, with the I or D allele of the Alu polymorphism occurring on diverse haplotype backgrounds [20]. The diversity of intragenic ACE haplotypes, their complex additive effects on ACE activity, and the observed inter-population differences [20] could account for the discrepant estimates of the proportion of the variance in ACE activity explained by the Alu polymorphism, where figures range from nil to 47% [9,11,12].

In agreement with previous studies, ACE activity measured in 92 Australian ADPKD patients was related to the I/D genotype, but the proportion of variance due to the Alu polymorphism was only 14%. A similar proportion can be assumed for the patients studied by Baboolal et al. [7], since they share the same ethnic background. The striking effect of the D allele on earlier development of renal failure reported in that study [7] is difficult to interpret: firstly, in view of the modest contribution of the Alu polymorphism to the control of ACE expression and secondly because of the lack of expected dosage effect of the D allele observed by Baboolal et al. [7] and Perez-Oller et al. [8]. Additionally our analysis of plasma ACE activity did not reveal any effect on the development of renal failure, in agreement with Uemaso et al. [9] and Van Dijk et al. [10].

In conclusion, the results from this study suggest that the ACE is not likely to play a role as a determinant of ADPKD phenotype severity. Unless information on the relation between the Alu polymorphism I/D genotypes and ACE activity is available, association studies are neither conclusive nor comparable.



   Acknowledgments
 
Funding for this work was provided by Edith Cowan University and the Medical Research Foundation of Royal Perth Hospital. We thank all ADPKD families for participating in the study and Dr Paul Burton for his assistance with the statistical analysis.



   Notes
 
Correspondence and offprint requests to: Mark Thomas, Department of Nephrology, Royal Perth Hospital, GPO Box X2213, Perth, Western Australia 6001, Australia. Email: Mark.Thomas{at}health.wa.gov.au Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 14. 4.01
Revision received 22. 6.01.