Angiotensin-converting enzyme activity and the ACE Alu ...

Nephrol Dial Transplant (2001) 16: 2323–2327

Original Article

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

Abstract Background. Previous studies concerning Alu IuD 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 IuD genotype may have confounded these studies. This investigation examines the association between ADPKD severity and ACE in terms of plasma enzyme activity and IuD 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 IuD 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 IuD genotype, showing a dosage effect of the D allele (Ps 0.006). The proportion of variance due to the Alu polymorphism was 14%. No difference in ACE activity and IuD genotype distribution was found between patients with CRF versus normal renal function (Ps 0.494; P s0.576) or between those with ESRF versus those without ESRF (P s 0.872; P s0.825). No effect of the

Correspondence and offprint requests to: Mark Thomas, Department of Nephrology, Royal Perth Hospital, GPO Box X2213, Perth, Western Australia 6001, Australia. Email: [email protected] #

IuD 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 Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by extensive inter- and intra-familial variation in clinical severity w1x. Since mutations in the PKD1 and PKD2 genes are the primary genetic cause of ADPKD, locus and mutation heterogeneity w2,3x, as well as the random nature of the somatic mutations proposed by the ‘‘two-hit’’ model w4x 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 w5x, 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 w6x. A number of studies have therefore

2001 European Renal Association–European Dialysis and Transplant Association

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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 w7,8x, and others failing to find an effect w9,10x. 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 w11,12x, and individual and population differences in the genetic control over ACE expression can be expected to have a confounding effect on studies where the IuD 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 IuD genotypes.

Subjects and methods 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 w13x. 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 mmolul (2.25 mgudl), 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 w14x. 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 w15x, 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 1.

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Methods Laboratory analyses. Plasma ACE activity was measured in 92 Australian patients using the kinetic method w16x, with a reference range of 23–100 Uul (covariance 3.2% at 125 Uul and 8.1% at 31 Uul). The ACE IuD polymorphism was detected by PCR amplification as described w17x. To avoid mistyping, all DuD homozygous samples were re-typed using an insertionspecific PCR primer w18x. 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 IuD 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 1ucreatinine 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 a of 0.05 (P-0.05). All analyses were performed using SPSS 9.0 (SPSS Inc, Chicago III).

Results The IuD polymorphism and plasma ACE activity In the overall group, the allele and genotype frequencies of the IuD polymorphism (Table 2) fell within the range reported previously in Caucasian populations w19,10x. A difference between the three populations was observed, with a lower frequency of the D allele in the Polish sample (P s 0.02). No deviation from HWE

Table 1. Characteristics of 307 ADPKD patients Country Australia Total # 116 Maleufemale 53 : 63 Genetic linkage PKD1 58 (50%) PKD2 3 (3%) Undefined 55 (47%) Mean current 46.6"1.4 age Hypertension 85 (73%) CRF 43 (37%) ESRF 33 (28%)

Overall Bulgaria

Poland

124 65 : 59

67 36 : 31

307 154 : 153

81 (65%) 27 (22%) 16 (13%) 46.7"1.5

45 (65%) 8 (12%) 14 (21%) 39.8"2.0

184 (60%) 38 (12%) 85 (28%) 45.2"0.9

65 (52%) 50 (40%) 26 (21%)

41 (61%) 24 (36%) 9 (13%)

192 (63%) 117 (38%) 68 (22%)

ACE activity and ACE Alu polymorphism

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Table 2. I/D genotypes observed in the three populations Country Australia Allele frequencies I 0.41 D 0.59 Genotypes IuI 21 (18%) IuD 54 (47%) DuD 41 (35%) Total 116

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

Bulgaria

0.41 0.59 13 (11%) 75 (61%) 36 (29%) 124

Renal function

Poland

0.52 0.48a 18 (27%) 34 (55%) 15 (22%) 67

0.45 0.55 52 (17%) 163 (53%) 92 (30%) 307

a The frequency of the D allele is significantly lower in the Polish group (P s 0.02).

was seen in the overall sample, nor in any individual population. Plasma ACE activity in all 92 Australian ADPKD patients fell within the reference interval (23–100 Uul). There was a significant correlation between Alu genotypes and enzyme activity, with a mean value of 41.2"5.2 Uul observed in IuI, 59.0"4.6 Uul in IuD, and 71.2"5.8 Uul in DuD subjects (P s 0.006). Similar results were obtained after adjustment for the use of ACE inhibitors in the control of hypertension (43.5" 6.5 Uul for IuI, 56.2"4.1 Uul for IuD and 69.7"4.5 Uul for DuD; P s 0.004). The fraction of the variance of plasma ACE activity explained by the ACE IuD 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 s 37; ESRF s27). The mean ACE activity levels were 61.7"3.8 Uul 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 s 0.494 for CRF vs normal renal function and P s 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 Uul in the group of patients reaching CRF before age 40 years (n s45) and 56.3"5.5 Uul among those reaching CRF after age 40 years. The difference was not significant (P s 0.352). The IuD genotype and ADPKD phenotype severity Risk of renal failure. The distribution of IuD genotypes among the 307 ADPKD subjects was examined by comparing subjects with normal renal function to those with CRF and with ESRF (Table 3). No significant differences were found between the three groups: P s 0.576 in the comparison of CRF vs normal renal function and P s 0.825 for ESRF vs lack of ESRF.

Normal CRF ESRF

ACE genotypes

Total

IuI

IuD

DuD

31 (16%) 21 (18%) 13 (19%)

97 (51%) 66 (56%) 35 (52%)

62 (32%) 30 (26%) 20 (30%)

190 117 68

P values are 0.576 for normal renal function vs CRF and 0.825 for ESRF vs lack of ESRF.

Age at development of renal failure. The relationship between the IuD 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 IuI, 39.7"1.1 for IuD and 39.1"1.5 for DuD subjects, Ps 0.810. The mean age at ESRF was 51.5" 3.0 years for IuI individuals, 55.5"1.6 for IuD and 54.5"1.7 for DuD, P s 0.415. This analysis was repeated after grouping the patients according to ethnic origin. Again, no statistically significant differences were found, with P s 0.739 and P s0.239 for the Australian patients, P s 0.504 and P s 0.087 for the Bulgarian and P s0.313 and P s 0.184 for the Polish. Lack of significant effect was observed when the different IuD genotypes were compared, within the entire group of patients using Kaplan–Meier cumulative survival curves to CRF (P s 0.815) and to ESRF (P s 0.478) (Figure 1). 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 IuD genotype on survival to CRF or ESRF. Hypertension. The distribution of IuD genotypes was compared between 192 hypertensive and 97 normotensive ADPKD subjects. The IuD distribution for the hypertensive group were: IuI (18%), IuD (51%), DuD (31%), and IuI (12.4%), IuD (57.7%) and DuD (29.9%) for the normotensive group. The analysis failed to reveal a significant difference between the two groups (P s 0.384).

Discussion The effect of the renin–angiotensin system and specifically of ACE activity on renal disease is an

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

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 IuD alleles and the ratio of PKD1 to PKD2 patients. However, none of the three populations displayed a significant association between the IuD 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 39 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 w20x. The diversity of intragenic ACE haplotypes, their complex additive effects on ACE activity, and the observed inter-population differences w20x 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% w9,11,12x. In agreement with previous studies, ACE activity measured in 92 Australian ADPKD patients was related to the IuD 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. w7x, since they share the same ethnic background. The striking effect of the D allele on earlier development of renal failure reported in that study w7x 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. w7x and Perez-Oller et al. w8x. Additionally our analysis of plasma ACE activity did not reveal any effect on the development of renal failure, in agreement with Uemaso et al. w9x and Van Dijk et al. w10x. 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 IuD genotypes and ACE activity is available, association studies are neither conclusive nor comparable. Acknowledgements. 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.

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