Pediatr Nephrol (2004) 19:734–737DOI 10.1007/s00467-004-1486-0

O R I G I N A L A R T I C L E

Hakan Erdogan · Sevgi Mir · Erkin Serdaroglu ·Afig Berdeli · Nejat Aksu

Is ACE gene polymorphism a risk factor for renal scarringwith low-grade reflux?

Received: 20 October 2003 / Revised: 11 March 2004 / Accepted: 12 March 2004 / Published online: 12 May 2004� IPNA 2004

Abstract The possible relationship between the angio-tensin-converting enzyme (ACE) gene insertion/deletion(I/D) polymorphism and renal scarring secondary tovesicoureteral reflux (VUR) has recently attracted atten-tion and the DD genotype was postulated to be a riskfactor for renal scarring. However, available data re-present conflicting results. The aim of this study was toinvestigate the ACE gene I/D polymorphism and the otherknown risk factors for renal scarring in children with low-and high-grade VUR. The study included 96 (67 females,29 males) patients (mean age at diagnosis 3.7€3.3 years)with VUR that were assessed for ACE I/D gene poly-morphism. ACE genotypes were determined as II, ID, andDD using the polymerase chain reaction. The controlgroup consisted of 103 healthy children with the sameethnicity to find the distribution of ACE gene I/D poly-morphism in the population. The frequency of renalscarring was 80.8% in the high-grade reflux group and34.3% in the low-grade reflux group. There was no dif-ference between groups with renal scarring and withoutscarring with respect to gender, family history of VUR,age at diagnosis of VUR, associated urological abnor-malities, frequency of urinary tract infection episodes, andthe occurrence of bilateral or unilateral VUR. GenotypeDD was found to be a significant risk factor for renalscarring in the study group by multivariate regression

analysis (odds ratio 3.79, P=0.011). It was not a riskfactor in high-grade reflux patients (odds ratio 0.60,P=0.62). However, it was a risk factor in low-grade pa-tients with respect to renal scarring (odds ratio 4.0,P=0.024). Although renal scarring is not common in low-grade reflux patients, there may be scarring in some pa-tients. DD polymorphism of the ACE gene is a significantrisk factor in low-grade reflux patients with renal scar-ring.

Keywords ACE gene polymorphism · Vesicouretericreflux · Renal scarring

Introduction

The most common cause of chronic renal failure (CRF) inTurkey is reflux nephropathy (RN) (32.4%) [1]. The eti-ology of RN and renal scarring is multifactorial. Thedegree of reflux, frequency of infection, age at diagnosis,delay in treatment of infection, and the nature of the in-fectious agents are the most important risk factors forrenal scarring [2, 3, 4]. The degree of reflux is a well-known risk factor. However, renal scarring may also de-velop in low-grade reflux, and is difficult to explain.

RN is a progressive process involving healing withfibrosis and focal segmental sclerosis. Genes controllingthe renin-angiotensin system play a role in this process offibrosis [5, 6, 7, 8, 9, 10]. Studies on angiotensinogen andangiotensinogen II type receptor gene polymorphism havefailed to show any association with renal fibrosis in RN.However, angiotensin-converting enzyme (ACE) inser-tion/deletion (I/D) gene polymorphism was found to havean effect on progression of IgA nephropathy [11], diabeticnephropathy [12], and polycystic kidney disease [13].Ozen et al. [5] showed the D allele to be an important riskfactor for the development of renal scarring in patientswith vesicoureteral reflux (VUR). Cho and Lee [6] andHazson et al. [7] also supported these findings. In con-trast, several investigators found that the D allele is not arisk factor for renal parenchymal damage [8, 9, 10].

H. Erdogan ()) · S. Mir · E. SerdarogluDepartment of Pediatric Nephrology,Ege University Medical School,35100 Izmir, Turkeye-mail: hakanerdogan66@yahoo.comTel.: +90-232-3885270Fax: +90-232-3885270

A. BerdeliDepartment of Pediatrics, Laboratory of Molecular Medicine,Ege University Medical School,Izmir, Turkey

N. AksuDepartment of Pediatric Nephrology,SSK Tepecik Teaching Hospital,Izmir, Turkey

The aim of this study was to investigate the ACE geneI/D polymorphism and other known risk factors for renalscarring in children with low- and high-grade VUR.

Patients and methods

The study included 96 Turkish children (67 females, 29 males),who were diagnosed with VUR by voiding cystourethrography(VCUG) at the Department of Pediatric Nephrology, Ege Univer-sity Medical School, between January 2000 and January 2003. Age-and sex-specific diagrams were used for calculating the bladdercapacity and for fluid filling volume of the bladder.

Patients who had secondary VUR due to neurogenic bladder orlower urinary system obstruction were excluded. The characteris-tics of the patients are presented in Table 1. The mean age atdiagnosis of VUR was 3.7€3.3 years (median 3 years) and the meanduration of follow-up was 3.8€2.9 years (median 3 years). Fifty-five patients had unilateral (33 left-sided, 22 right-sided) and 41patients had bilateral VUR. The VCUG findings were graded fromgrade I to grade V according to the International Reflux Classifi-cation [14]. VUR grades I–V were 4.2%, 27.1%, 41.7%, 19.8%,and 7.3%, respectively. Patients were divided into two groups ac-cording to their VUR grade: low (grade I, II, and III) and high(grade IV and V). A family history of VUR, number of urinaryinfection episodes, associated urological abnormalities, and de-mographic data were recorded.

99mTc-DMSA scanning was performed for all the patientswithin 4–6 months of diagnosis of VUR. Posterior, anterior,and bilateral oblique images were taken 3 h after injection of99mTc-DMSA. Abnormal 99mTc-DMSA scans were repeated after6 months and these images were compared with the initial images.Renal scarring was diagnosed as a focal or generalized uptakedefect and/or renal contour defect observed in both of the subse-quent investigations.

Genomic DNA was extracted from the EDTA-treated blood.ACE gene I/D polymorphism was detected with PCR using specificPCR probes and the DD genotype was verified by a second PCR.The control group consisted of 103 healthy children with the sameethnicity to determine the distribution of ACE gene I/D polymor-phism in the population. The families of the patients and controlgroup gave their written informed consent to the study, which wasapproved by the medical faculty ethics committee of our university.

Determination of ACE genotypes

Genomic DNA was prepared from whole blood using QiAampDNA Blood Mini kit (QIAGEN, Hilden, Germany) according to themanufacturer’s recommendations.

A 50-ng sample of genomic DNA was amplified by PCR (GeneAmp PCR System 9700, PE Applied Biosystems) in a 25-�l mix-ture of 1x Gene Amp PCR Buffer II with 1.5 mM MgCl2 (AppliedBiosystems, Branchburg, N.J., USA), 200 �M of each deoxynu-cleotide triphosphate (dNTP)(Promega, Madison, Wis., USA),5 pmol sense and antisense primers, 0.2 �l AmpliTaq DNA Poly-merase (Applied Biosystems, 5 U/�l), and nuclease-free water. Thesequences of the sense and antisense primers were 50-CTGGA-GACCACTCCCATCCTTTCT-30 and 50-GATGTGGCCATCA-CATTCGTCAGAT-30 (TIB MOLBIOL Syntheselabor, Berlin,Germany), respectively. Thermal cycling was carried out at 94�Cfor 10 min, then 32 cycles of 94�C for 30 s, annealing at 58�C for50 s and extension at 72�C for 60 s, followed by 7 min of finalextension at 72�C.

The PCR products were separated by 2% agarose gel electro-phoresis, and 490 bp with insertion (I allele) and 190 bp withdeletion (D allele) were visualized with ethidium bromide stainingin the UVP BioDoc-It System Biolmaging Systems (Upland, Calif.,USA).

To avoid the possibility of mistyping the ID heterozygotes asDD homozygotes, all DD samples were reamplified with a second

primer pair specific for the inserted sequence intron 16 of the ACEgene. PCR was performed in a final 25-�l mixture of 1.5 mMMgCl2, 20 pmol primers, 2 U Taq polymerase, and 100 ng genomicDNA. The primers for the inserted sequence were 50-TGGGAC-CACAGCGCCCGCCACTAC-30 and 50-TCGCCAGCCCTCC-CATGCCCATAA-30 (TIB MOLBIOL Syntheselabor).

Thermal cycling was carried out at 94�C for 10 min, then 30cycles with 30 s denaturation at 94�C, followed by 30 s annealing at67�C, extension for 30 s at 72�C, and 10 min final extension at72�C. The PCR products were separated by 2% agarose gel elec-trophoresis; the 335-bp DNA fragment with ethidium bromidestaining was visualized in the UVP BioDoc-It System BiolmagingSystems. Only the I allele produced a 335-bp fragment, while noproducts were detected with the DD genotype, which assured theabsence of mistyping.

Statistical methods

The data were analyzed using SPSS for Windows 9.05 software.The differences in numerical variables between groups were ana-lyzed by Mann-Whitney U test. The association between the groupswas analyzed using chi-squared and Fischer exact test when fea-sible. Odds ratio and 95% confidence intervals (CI) were used inmultiple logistic regression analysis for the assessment of riskfactors. The multiple logistic regression analysis was also used fordefining risk factors. A P value less than 0.05 and an odds ratiowith 95% of confidence not including 1 was considered as statis-tically significant.

Results

Forty-five patients (46.9%) had renal scarring (25 patientsunilateral, 20 patients bilateral) and 51 patients (53.1%)had no scar (30 patients unilateral, 21 patients bilateral)with subsequent 99mTc-DMSA scanning. ACE gene I/Dpolymorphism and allele frequency are shown in Table 2in VUR patients with and without renal scarring and thecontrol group.

High-grade reflux was more frequent in patients withrenal scarring than in patients without renal scarring whenall patients (n=96) were evaluated by one-way analysis(46.7% vs. 9.8%, odds ratio 8.05, 95% CI 2.69–24.01,P<0.0001). The frequency of renal scarring was 80.8% inthe group with high-grade reflux and 34.3% in the groupwith low-grade reflux. There were 24 patients with renalscarring in the group with low-grade VUR. Although theurinary tract infection episodes were more frequent in

Table 1 Characteristics of the 96 children with vesicoureteral re-flux (VUR) (UTI urinary tract infection)

Female/male 67/29Age at diagnosis (years) 3.73€3.39Age at diagnosis (<1 year) 24 (%25)Follow-up time (years) 3.89€2.91Low-grade VUR 70 (72.9%)Grade I 4 (4.2%)Grade II 26 (27.1%)Grade III 40 (41.7%)High-grade VUR 26 (27.1%)Grade IV 19 (19.8%)Grade V 7 (7.3%)UTI frequency 2.96€2.75Renal scarring 45 (46.9%)

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patients with renal scarring, this was not statisticallysignificant (3.3€2.5 vs. 2.6€2.9, P=0.0578). There was nodifference between the groups with and without renalscarring with respect to gender, family history of VUR,age at diagnosis of VUR, associated urological abnor-malities (5 patients with duplication of urinary system, 1patient with horseshoe kidney, 2 patients with solitarykidney), and the occurrence of bilateral or unilateralVUR. Risk factors in the patient population were ana-lyzed by multiple logistic regression analysis (Table 3).The D allele frequency was found to be a risk factor forrenal scarring in the study group by multivariate regres-sion analysis (odds ratio 3.79, P=0.011).

Since the grade of VUR was the most important riskfactor for the development of renal scarring (odds ra-tio=16.55) and there was a high prevalence of renalscarring in patients with low-grade VUR (34.3%), riskfactors in high- and low-grade VUR were evaluatedseparately. In the group with high-grade reflux, all theentities, including D allele frequency, were not found tobe significant for renal scarring.

The important factors for renal scarring were evaluatedby multiple logistic regression analysis for the group withlow-grade reflux (Table 4). The D allele was found to bethe most important risk factor for the development ofrenal scarring in the group with low-grade VUR (oddsratio 3.93, 95% CI 1.15–13.42, P=0.028). Other factors

were not important in the development of renal scarring inthe group with low-grade VUR.

Discussion

The grade of VUR, age at diagnosis, the number of re-current urinary tract infections, delay in initiating treat-ment, and the nature of the infectious agent are well-known important risk factors for the development of renalscarring in VUR. In recent years, several studies haveinvestigated gene polymorphism affecting renal fibrosis.The ACE gene I/D polymorphism is the most-studied riskfactor for renal scarring in VUR; however, the results arestill controversial [5, 6, 7, 8, 9, 10].

Previous studies have reported that the DD genotype ofthis polymorphism is associated with higher levels ofplasma and tissue ACE activity. This genetic increase inACE activity may result in greater angiotensin II forma-tion in renal tissue [15, 16]. Angiotensin II increases theintraglomerular pressure, inducing the transforminggrowth factor to exert a prosclerotic activity. This leads tointerstitial proliferation and activation of the plasminogenactivator inhibitor, which prevents the degradation of theglomerular interstitium. Glomerular sclerosis is thus ag-gravated [17].

In our study, the frequency of the D allele was higherin the group with scarring than the group without scarring,but the difference was not statistically significant by one-way analysis. Nevertheless, a result that was close to thelimit of significance was obtained. High frequency of theD allele increased the scarring 3.79 times by multivariateanalysis.

In addition, the D allele was more frequent in thegroup with renal scarring, but not the group withoutscarring, compared with controls. Ozen et al. [5] reportedsimilar results in a study of Turkish children. Cho and Lee[6] and Haszon et al. [7] also reported the D allele to be arisk factor in the development of renal scarring in patientswith VUR. In contrast, Dudley et al. [8] reported the Dallele to be a significant risk factor for the development ofhypertension but not for scarring in patients with VUR.Ohtomo et al. [9] reported that the D allele was frequentlyfound in patients with VUR but this frequency was notstatistically significant. A recent study by Pardo et al. [10]

Table 2 Angiotensin-converting enzyme insertion/deletion (I/D)polymorphism and allele frequencies of patients with and withoutrenal scarring with VUR and the healthy control group (one-wayanalysis)

ACEgenotypes

Renal scar (+) Renal scar (�) Controlgroup

n=45 (%) n=51 (%) n=103 (%)

II genotype 2 (4.4) 4 (7.8) 20 (19.4)ID genotype 21 (46.7) 30 (58.8) 56 (54.4)DD genotype 22 (48.9) 17 (33.3) 27 (26.2)D allele*4 72%*1,*3 63%*1,*2 53%*2,*3

*1 Renal scar (+) vs. renal scar (�), P=0.0719*2 Renal scar (�) vs. control group, P=0.0750*3 Renal scar (+) vs. control group, P=0.0004*4 All VUR patients vs. control group, P=0.0012

Table 3 Evaluation of risk factors for renal scarring in childrenwith VUR by multivariate logistic regression analysis (n=96)(OR odds ratio, CI confidence interval)

Risk factors P r OR 95% CI

Male sex 0.789 0.000 1.80 0.34–3.99Positive familyhistory for VUR

0.469 0.000 1.98 0.31–12.67

Age at diagnosis 0.659 0.000 1.03 0.89–1.20Additional urologicalabnormalities

0.273 0.000 2.08 0.56–7.72

Bilateral VUR 0.287 0.000 0.56 0.19–1.61UTI episodefrequency

0.110 0.066 1.16 0.96–1.39

D allele frequency 0.011 0.186 3.79 1.34–10.72VUR grade 0.001 0.339 16.55 4.24–64.46

Table 4 Evaluation of risk factors for renal scarring in childrenwith low-grade VUR by multivariate logistic regression analysis(n=70)

Risk factors P r OR 95% CI

Male sex 0.308 0.000 2.40 0.44–13.00Positive family historyfor VUR

0.587 0.000 2.03 0.15–26.25

Age at diagnosis 0.141 0.043 1.13 0.95–1.35Additional urologicalabnormalities

0.392 0.000 2.09 0.38–11.34

Bilateral VUR 0.175 0.000 0.41 0.11–1.47UTI episode frequency 0.117 0.072 1.16 0.96–1.42D allele frequency 0.028 0.181 3.93 1.15–13.42

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also reported that the frequency of the D allele had noeffect on the development of renal scarring.

It is well known that renal scarring is not an expectedfeature in patients with low-grade reflux. The grade ofVUR was the most important risk factor for renal scar-ring, as shown in our study. However, renal scarring de-velops in some of these patients and the classical riskfactors do not account for this renal scarring. About 73%of the patients in our study had low-grade reflux, and 34%of these had renal scar formation. When these patientswere evaluated for the risk factors for renal scarring, ACEgene DD genotype was the single risk factor. Dudley et al.[8] and Pardo et al. [10] divided their patients accordingto VUR grade (grade I–II and III–V and grade I–III andgrade IV–V, respectively). They reported that ACE geneDD polymorphism was not a risk factor for renal scarringin low-grade VUR, in contrast to our study [8, 10]. In astudy conducted by Ozen et al. [5] with patients of thesame ethnicity as our study group, grade I and II refluxpatients were excluded and ACE gene DD genotype wasnot studied in patients with low-grade reflux.

In conclusion, the ACE gene I/D polymorphism Dallele is more frequently seen in VUR patients thancontrols and the D allele is a significant risk factor forrenal scarring. We hypothesize that this significanceoriginates from the effect of the D allele on low-gradeVUR. Patients with low-grade reflux and with ACE DDgenotype should be considered at risk of scarring. Closefollow-up and treatment of these patients may reduce themorbidity.

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