LOW RENIN-ANGIOTENSIN SYSTEM ACTIVITY GENE POLYMORPHISMAND DYSPLASIA ASSOCIATED WITH POSTERIOR URETHRAL VALVES

LICIA PERUZZI,* FEDERICA LOMBARDO, ALESSANDRO AMORE, EMILIO MERLINI,GABRIELLA RESTAGNO, LEANDRA SILVESTRO, TERESA PAPALIA AND ROSANNA COPPO

From the Nephrology, Dialysis and Transplantation Unit (LP, AA, RC), and Department of Molecular Genetics (FL, GR), ReginaMargherita Children’s Hospital, Department of Pediatric Surgery, Maggiore Hospital Novara (EM) and Pediatric Clinic, University of

Turin (LS), Turin and Nephrology, Dialysis and Transplantation Unit, S. Annunziata Hospital, Cosenza (TP), Italy

ABSTRACT

Purpose: Obstructive uropathies, including posterior urethral valves (PUVs) and kidney hypo-dysplasia, are the most frequent cause of renal failure in children. The role of renin-angiotensinsystem genes in renal and urinary tract development has been observed in experimental models.The aim of this study was to investigate the distribution of angiotensin converting enzyme (ACE),angiotensinogen (AGT) and angiotensin receptor type 1 (ATR1) genetic polymorphisms in chil-dren affected by chronic renal failure due to renal hypodysplasia associated with posteriorurethral valves or without urethral abnormalities.

Materials and Methods: The study included 50 children (21 with hypodysplasia associated withPUVs, 7 with obstructive uropathy and 22 with pure hypodysplasia) and 50 healthy subjectsmatched for sex and geographic origin. ACE ID, AGT TC and ATR1 AC gene polymorphisms wereassayed in all patients with standard polymerase chain reaction techniques.

Results: ACE II was expressed more in patients with PUVs compared to those with otherdysplasias and controls (43% vs 7% and 10%, respectively, chi-square test p �0.05), while ATR1AA was significantly less represented in patients with hypodysplasia compared to controls (38%vs 56%, chi-square test p �0.05). ACE DD and AGT genotypes were not distributed differently inpatients with PUVs compared to those with other dysplasias and controls.

Conclusions: To our knowledge this is the first report associating severe congenital uropathiesand renal hypodysplasia with decreased renin-angiotensin system activity associated with theACE II genotype and a possible functional imbalance among ATR1 receptors.KEY WORDS: renin-angiotensin system; polymorphism, genetic; kidney failure, chronic; urethra; urogenital abnormal-

ities

Obstructive uropathies, including posterior urethral valves(PUVs) and kidney hypodysplasia, are the most frequentcause of end stage renal disease in children.1 Human obser-vations2, 3 and experimental models4 indicate the strict asso-ciation between PUVs and renal parenchymal dysplasia asindependent primary malformations or as related defects, egdysplasia due to early urethral obstruction.

Among large series of genes the renin-angiotensin system(RAS) has a pivotal role in regulating renal developmentfrom the early stages of pregnancy.5, 6 RAS genetic or phar-macological impairment produces in offspring significant re-nal abnormalities of vascular, interstitial and tubular struc-tures, as well as a variety of urinary tract malformations.7�10

RAS activity is genetically modulated as angiotensin convert-ing enzyme (ACE) and angiotensinogen (AGT) gene polymor-phisms influence enzyme serum levels, and angiotensin re-ceptor type 1 (ATR1) affects signal transduction.

Insertion/deletion polymorphism of a fragment of intron 16of the ACE gene is responsible for 50% of the variability of

serum enzyme levels found in the population. The I allele isassociated with lower enzyme activity.11

T704C AGT gene polymorphism, which causes a transitionat nucleotide 704 with substitution of methionin with threo-nine, causes decreased enzyme activity in threonine/threo-nine (CC) homozygous subjects.12 ATR1 has its functionalactivity related to the 1166 AC polymorphism, with loweractivity due to homozygous AA genotype.13

We sought to investigate the distribution of ACE, AGT andATR1 gene polymorphisms in a highly selected series ofchildren affected by chronic renal failure detected in the firstyear of life due to renal dysplasia associated with PUVs orwithout urethral abnormalities.

MATERIALS AND METHODS

Patients. Patients or parents of subjects younger than 18years gave written informed consent. Selection criterion wasdecreased renal function (glomerular filtration rate[Schwarz’s formula] less than 75 ml per minute per 1.73 m2)due to congenital nephropathy with bilaterally reduced renalmass diagnosed within the first year of life by renal ultra-sound and scintigraphy with 99mtechnetium dimercapto-succinic acid (defined herein as hypodysplasia). Renal biopsyto confirm dysplasia was not performed, since it was notconsidered ethically appropriate. Hence, the term dysplasiais adopted as the wider exception of reduced renal massassociated with defective function due to abnormal organo-genesis.

A total of 50 children (40 males and 10 females) fulfilling

Submitted for publication December 1, 2004.Study fulfilled standards of the Declaration of Helsinki, and re-

ceived regional ethical committee approval.Supported by a grant from Fondazione Piera, Pietro e Giovanni

Ferrero and by a grant from the Italian Ministry of University andScientific Research. The study was carried out within the doctorateof philosophy course in experimental pediatrics at the University ofTurin, Turin, Italy.

* Correspondence: Nephrology, Dialysis and Transplantation, Re-gina Margherita Children’s Hospital, Piazza Polonia 94, 10126Torino, Italy (telephone: 39–011-3135362; FAX: 39–011-6635543;e-mail: rene@oirmsantanna.piemonte.it).

0022-5347/05/1742-0713/0 Vol. 174, 713–717, August 2005THE JOURNAL OF UROLOGY® Printed in U.S.A.Copyright © 2005 by AMERICAN UROLOGICAL ASSOCIATION DOI: 10.1097/01.ju.0000164739.13408.e2

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study criteria were selected. Of these patients 21 had renalhypodysplasia associated with PUVs, 7 had renal hypodys-plasia associated with other obstructive uropathies (uretero-pelvic junction stenosis, ureterovesical junction stenosis) and22 had pure renal hypodysplasia without urological malfor-mations. A total of 31 children were followed for more than 5years. End stage renal failure developed in 19 patients (11with PUVs), with the requirement for substitutive therapyduring childhood (9 children before age 5 years).

Controls. The control group consisted of 50 healthy sub-jects with analogous distribution for sex, age and geographicorigin.

Genetic analysis. Genomic DNA was extracted from peripheralblood mononuclear cells using a DNA isolation kit. ACE ID poly-morphism was typed with polymerase chain reaction (PCR) using25 pmol primer set 5�-CTGGAGACCACTCCCATCCTTTCT-3�and 25 pmol 5�-GATGTGGCCATCACATTCGTCAGAT-3� in avolume of 50 �l, 2.5 mm magnesium chloride, 0.2 mm D-nucleotidetriphosphates, 0.2 mm dimethyl sulfoxide 5% and 2.5 units Taqpolymerase. DNA was amplified by 30 cycles at 94, 58 and 72degrees for 1 minute each. Extension was for 10 minutes at 72degrees. PCR products were separated on 2% agarose gel stainedwith ethidium bromide, and all DD genotypes were checked withI specific primers to exclude the mistyping of the ID type as the DDtype (5�-TGGGACCACAGCGCCCGCCACTAC-3� and 5�-TCG-CCAGCCCTCCCATGCCCATAA-3�).

AGT polymorphism. The T704C variant sequence of the AGTgene was amplified by PCR using 25 pmol primer set 5�-CAGGCTGTGACAGGATGGAAGACTGGCTGCTCGTTGA-3�and 25 pmol 5�-CTGCCCATCTCCAAGGCCTGACTG-3� in 30�l containing 2.5 mm magnesium chloride, 0.2 mmD-nucleotide triphosphates and 2.5 units Taq polymerase. Cy-cling conditions were denaturation at 95 degrees for 5 minutes,32 cycles of 45 seconds at 94 degrees, 45 seconds at 68 degreesand 1 minute at 72 degrees, and final extension at 72 degreesfor 7 minutes. The PCR product was digested with 5 units HincII restriction enzyme at 37 degrees nightly. Fragments wereseparated as described previously.

ATR1 polymorphism. The 1166 AC polymorphism in the 3�region of the ATR1 gene was examined with 25 pmol primer5�-AGAAGCCTGCACCATGTTTTGAG-3� and 25 pmolprimer 5�-CCTGTTGCTCCTCTAACGATTTA-3� by PCR in atotal of 30 �l. Initial denaturation was at 95 degrees for 5minutes, followed by 35 cycles of 30 seconds at 94 degrees, 30seconds at 57 degrees and 30 seconds at 72 degrees, and finalextension at 72 degrees for 7 minutes. The 410 base pairamplified fragment was digested with 5 units DdeI at 37degrees nightly and visualized on gel as described previously.

Statistical analysis. Comparison among genotype frequen-cies was done using the chi-square test based on 2 � 2 tables.Potential associations between genotypes and diagnosis werefirst tested using the univariate method (chi-square test andodds ratio [OR], 95% confidence interval [CI]), then by mul-tivariate analysis. Multivariate logistic regression analysiswas used to assess the predictive value of polymorphisms

related to PUVs. A p value of less than 0.05 was consideredstatistically significant. SPSS® 8.0 software was used forstatistical analyses.

RESULTS

ACE genotype distribution. The II genotype was signifi-cantly more represented in patients with renal hypodyspla-sia and early end stage renal failure than in controls. Of the50 children with hypodysplasia 11 (22%) had the II genotypevs 5 (10%) in the control group (chi-square test 5.36, p � 0.02,OR � 2.53, 95% CI 1.06 to 6.15, table 1, and figs. 1 and 2).

There were no differences from the control population forthe DD genotype (15 patients, or 30%, vs 17 controls, or 34%,chi-square test 0.37, p � 0.54 [not significant], OR 0.83, 95%CI 0.43 to 1.57) or the ID genotype (24 patients, or 48%, vs 28controls, or 56%, chi-square test 1.28, p � 0.25 [not signifi-cant], table 1, and figs. 1 and 2). The allele frequency for I andD was similar in patients and controls (I 46% vs 38%, chi-square test 1.31, p � 0.25 [not significant], and D 54% vs62%, chi-square test 1.31, p � 0.25 [not significant]).

In the PUVs group, which was highly selected for theseverity of renal dysplasia, the ACE II genotype was signif-icantly more frequent (9 of 21 patients, or 43%) compared tocontrols (5 of 50 patients, or 10%, chi-square test 27.95,p �0.0001, OR 6.78, 95% CI 2.99 to 15.72, table 2 and fig. 3).The data were similar when the control population was in-creased to 100 healthy subjects, with ACE II present in 43%of the PUVs group vs 14% of controls (chi-square test 16.7,p �0.0001, OR 3.94, 95% CI 1.89 to 8.28).

ACE II genotype distribution was also more frequent inpatients with PUVs (9 of 21 patients, or 43%) than in thosewithout PUVs (2 of 29 patients, or 7%, chi-square test 34.5,p �0.0001, OR 10, 95% CI 3.98 to 26.29, table 2, and figs. 3and 4). Similar results were obtained regarding the I allele,which in patients with PUVs had a frequency of 57% com-pared to 38% in those without PUVs and 38% in controls(chi-square test 7.23, p � 0.0071, OR 2.16, 95% CI 1.18 to3.96).

The ACE DD genotype in our series was not differentlydistributed between controls and patients with hypodyspla-sia. Among 50 children with hypodysplasia 15 (30%) were DDpositive vs 17 controls (34%, chi-square test 0.36, p � 0.37[not significant], OR 0.83, 95% CI 0.43 to 1.57, table 1).Similarly, the ACE DD genotype was not differently distrib-uted between the subgroup of patients with PUVs comparedto controls. Among 21 patients with PUVs 6 (29%) were DDpositive compared to 17 controls (34%, chi-square test 0.57,p � 0.37 [not significant], OR 0.79, 95% CI 0.417 to 1.50).Likewise, of the 21 patients with hypodysplasia withoutPUVs 6 (29%) were DD positive vs 9 of 29 controls (31%,chi-square test 0.09, p � 0.75 [not significant], OR 0.90, 95%CI 0.47 to 1.74, table 2 and fig. 3). Multivariate crosstabsconsidering the 3 genotypes in patients with and withoutPUVs and controls confirmed the results obtained with uni-

TABLE 1. Genotype frequency in patients with hypodysplasia and controls

No. Hypodysplasia (%) No. Controls (%) Chi-Square Test p Value OR (95% CI)

No. pts 50 50ACE genotypes:

II 11 (22) 5 (10) 5.36 0.02 2.53 (1.06–6.15)ID 24 (48) 28 (56) 1.28 0.25 0.725 (0.39–1.31)DD 15 (30) 17 (34) 0.37 0.54 0.83 (0.43–1.57)

AGT genotypes:CC 19 (38) 16 (32) 0.79 0.37 1.30 (0.69–2.43)TC 23 (46) 26 (52) 0.72 0.39 0.78 (0.43–1.42)TT 8 (16) 8 (16) 0.00 1.00 1 (0.44–2.27)

ATR1 genotypes:AA 24 (48) 28 (56) 1.28 0.25 0.72 (0.39–1.31)AC 23 (46) 16 (32) 4.11 0.04 1.81 (0.97–3.35)CC 3 (6) 6 (12) 2.19 0.13 0.46 (0.14–1.41)

LOW RENIN-ANGIOTENSIN SYSTEM ACTIVITY GENE POLYMORPHISM AND DYSPLASIA714

variate analysis (Pearson chi-square test 15.78, p � 0.004,with II genotype as the driving factor).

AGT genotype distribution. Patients with renal dysplasiadid not demonstrate a significant difference in AGT genotype

frequency compared to controls. CC was found in 19 of 50patients with hypodysplasia (38%) vs 16 of 50 controls (32%,chi-square test 0.79, p � 0.37 [not significant], OR 1.30, 95%CI 0.69 to 2.43), TC in 23 patients with hypodysplasia (46%)vs 26 controls (52%, chi-square test 0.72, p � 0.39 [not sig-nificant], OR 0.78, 95% CI 0.43 to 1.42) and TT in 8 patientswith hypodysplasia (16%) vs 8 controls (16%, chi-square test0.00, p � 1.0, OR 1, 95% CI 0.44 to 2.27, table 1 and fig. 1).Also, in patients with PUVs none of the AGT genotypesdisplayed a distribution different from patients withoutPUVs or controls (table 2 and fig. 3).

Allelic distribution in patients with hypodysplasia did notdiffer from controls (C 62% vs 58%, T 38% vs 42%, notsignificant). Even disease subgrouping did not show signifi-cant differences in allelic distributions in patients with ver-sus without PUVs and healthy controls.

ATR1 genotype distribution. The total cohort of patientswith renal hypodysplasia did not exhibit a significant differ-ence in ATR1 genotype distribution compared to controls. AAwas present in 24 of 50 patients with renal hypodysplasia(48%) vs 28 of 50 controls (56%, chi-square test 1.28, p � 0.25[not significant], OR 0.725, 95% CI 0.399 to 1.31), AC in 23patients (46%) vs 16 controls (32%, chi-square test 4.11,p � 0.04, OR 1.81, 95% CI 0.978 to 3.35) and CC in 3 patients(6%) vs 6 controls (12%, chi-square test 2.19, p � 0.13 [notsignificant], OR 0.46, 95% CI 0.48 to 1.41, table 1 and fig. 1).

Disease subgrouping revealed that AA genotype was sig-nificantly less represented in patients with PUVs comparedto those without PUVs and controls. AA was present in 8 of21 patients with PUVs (38%) vs 28 of 50 controls (56%,chi-square test 6.50, p � 0.01, OR 0.48, 95% CI 0.26 to 0.88)and vs 16 of 29 patients without PUVs (55%, chi-square test5.80, p � 0.01, OR 0.50, 95% CI 0.27 to 0.91, table 2). The CCgenotype was not distributed differently between patientswith PUVs (2 of 21, or 10%) and controls (6 of 50, or 12%,chi-square test 0.54, p not significant, OR 0.70, 95% CI 0.25to 1.91) or patients without PUVs (1 of 29, or 3%, chi-squaretest 3.06, p � 0.08, OR 3.12, 95% CI 0.74 to 11.22, table 2 andfig. 3). The same trend of lower ATR1 A allele distributionwas observed in patients with PUVs, although it did notreach statistical significance (A 64% in patients with PUVsvs 76% in those without PUVs, chi-square test 3.42,p � 0.06).

Association of genotypes with low RAS. Logistic regressionanalysis considering PUVs and putative risk factors assumedas genotypes determining low RAS activity (ACE II, ATR1AA and AGT CC) assigned a strong predictive value to PUVsfor the ACE II genotype (chi-square test 15.097, p � 0.001,ACE II with B 2.10 � 064, p � 0.0011, R 0.2914, Exp [B]8.23). A lower frequency of the ATR1 AA genotype was also arisk factor for PUVs, although these data did not reach sta-tistical significance (B -1.01 � 0.59, p � 0.08, R -0.095,Exp [B] 0.36).

DISCUSSION

Angiotensin II, besides being an inducer of sclerogenousgene expression14 and being involved in the progression ofrenal diseases, is a powerful growth factor whose expressionduring embryogenesis is fundamental to appropriate devel-opment, particularly in high angiotensin II receptor express-ing organs such as the kidney. These effects were observed inexperimental models of RAS blockade at different levels andwere attributed to the lack of the growth promoting action ofangiotensin II.7�10 In recent years it was clarified that thecontacts between renal mesenchyma and ureteral bud areregulated by a complex sequence of induction, proliferationand apoptosis, with timely expression of a series of genes,including those of the RAS.8, 15 These experimental resultsare also supported in humans by the observation of renal

FIG. 1. ACE ID, ATR1 A1166C and AGT (Met235-Thr) genotypefrequency in entire cohort of patients with hypodysplasia and incontrols.

FIG. 2. Odds ratio for development of renal hypodysplasia com-pared to controls for ACE genotypes II, DD and ID with 95% confi-dence interval.

LOW RENIN-ANGIOTENSIN SYSTEM ACTIVITY GENE POLYMORPHISM AND DYSPLASIA 715

teratogenic effects induced by pharmacological blockade ofRAS during pregnancy.16

Moreover, angiotensin II effects are dependent on the typeof receptor involved. ATR1 in fetal life is mainly expressed inthe deeper areas of the developing kidney, namely glomeruli,

mesangial and juxtaglomerular cells, where it seems to havea role in renovascular development and glomerulogenesis,while ATR2 is expressed in the ureteral bud and seems tocoordinate the mesenchymal cell turnover and contacts withthe ureteral bud.7, 8, 17 These differential expressions mayexplain why in experimental models parenchymal abnormal-ities were mainly associated with the lack of AGT, ACE andATR1, while the absence of ATR2 particularly correlated toureteral malformations.

TABLE 2. Genotype frequency in patients with and without PUVs, and controls

No. WithPUVs (%)

No. Controls(%)

Chi-SquareTest p Value OR (95% CI) No. Without

PUVs (%)Chi-Square

Test p Value OR (95% CI)

No. pts 21 50 29ACE genotypes:

II 9 (42.9) 5 (10) 27.95 �0.0001 6.78 (2.99–15.72) 2 (6.9) 34.5 �0.0001 10 (3.98–26.3)ID 6 (28.6) 28 (56) 14.9 0.0001 0.32 (0.17–0.59) 18 (62.1) 27.4 �0.0001 0.21 (0.11–0.39)DD 6 (28.6) 17 (34) 0.57 0.37 0.79 (0.417–1.50) 9 (31) 0.095 0.75 0.90 (0.47–1.74)

AGT genotypes:CC 9 (42.9) 16 (32) 2.58 0.10 1.6 (0.86–2.97) 10 (34.5) 1.71 0.19 1.46 (0.79–2.70)TC 8 (38.1) 26 (52) 3.96 0.05 0.56 (0.30–1.03) 15 (51.7) 3.95 0.04 0.56 (0.30–1.03)TT 4 (19) 8 (16) 0.311 0.57 1.23 (0.55–2.72) 4 (13.8) 0.91 0.34 1.44 (0.63–3.27)

ATR1 genotypes:AA 8 (38.1) 28 (56) 6.5 0.01 0.48 (0.26–0.88) 16 (55.2) 5.8 0.01 0.50 (0.27–0.91)AC 11 (52.4) 16 (32) 8.21 0.004 2.3 (1.2–4.2) 12 (41.4) 2.43 0.11 1.55 (0.85–2.83)CC 2 (9.5) 6 (12) 0.54 0.70 0.70 (0.25–1.91) 1 (3.4) 3.06 0.08 3.12 (0.74–11.22)

FIG. 3. ACE, ATR1 and AGT genotype distribution in patientswith (PUV) vs without posterior urethral valves (non-PUV).

FIG. 4. Odds ratio with 95% confidence interval for ACE genotypeII for patients with (PUV) vs without posterior urethral valves (non-PUV) and controls.

LOW RENIN-ANGIOTENSIN SYSTEM ACTIVITY GENE POLYMORPHISM AND DYSPLASIA716

The data gathered in this study demonstrate a significantassociation between the ACE genotype depressing RAS ac-tivity and severe renal hypodysplasia associated with PUVs.A role of ACE ID polymorphism favoring increased enzymetissue concentration has been observed in the progression ofmost adult nephropathies,18 as well as in some unselectedseries of childhood uropathies,19, 20 while its role in renalabnormal organogenesis has not been described.

The data derived from this study, in a highly homogeneousand strongly negatively selected series of children affected byearly end stage renal failure due to hypodysplasia, supportthe hypothesis of a genetically determined “low renin angio-tensin environment” as a possible contributing factor to renalhypodysplasia associated with PUVs. The ACE genotype II,conditioning a lower ACE activity, was observed significantlymore frequently in patients with renal hypodysplasia than incontrols. The II genotype in our series was particularly rep-resented in the PUVs group, with a highly significant oddsratio. Moreover, multivariate analysis identified the ACE IIgenotype as the only genetic risk factor for hypodysplasiaassociated with PUVs.

In the same population of patients affected by PUVs adecreased frequency of ATR1 AA genotype, conditioninglower receptor activity, was also found. This condition mayresult in a loss of balance between the 2 functional angioten-sin II receptors, ATR1 and ATR2, with a shift toward in-creased ATR1 activity and parallel decrease in ATR2 relatedeffects, in agreement with experimental data in animals.6�10

The mechanism of development of PUVs has not beenclarified in its molecular steps thus far. The low renin-angiotensin environment observed in this study may have acrucial effect on the development of kidney hypodysplasiaand on the derangement of the separation process of rectumfrom the urogenital sinus, with persistence of plicae of meso-nephric ducts giving rise to the urethral valves.

CONCLUSIONS

To our knowledge this study shows for the first time anassociation between RAS gene polymorphisms conditioninglow angiotensin II environment and congenital nephropa-thies presenting with renal mass reduction and early onsetchronic renal failure. The association was particularly strongin children with PUVs, although the pathogenic mechanismremains to be defined.

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