J Am Soc Nephrol 11: , 2000

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1 J Am Soc Nephrol 11: , 2000 Polymorphism of Angiotensin Converting Enzyme, Angiotensinogen, and Angiotensin II Type 1 Receptor Genes and End-Stage Renal Failure in IgA Nephropathy: IGARAS A Study of 274 Men LUC FRIMAT,* CHRISTOPHE PHILIPPE, MARIE-NOËLLE MAGHAKIAN,* PHILIPPE JONVEAUX, BRUNO HURAULT DE LIGNY, FRANCIS GUILLEMIN, and MICHÈLE KESSLER* Departments of *Nephrology, Genetics, and Biostatistics, University Hospital, Nancy; Department of Nephrology, University Hospital Caen, France. Abstract. The impact of renin-angiotensin system (RAS) gene polymorphism on the prognosis of IgA nephropathy (IgAN) is still debated. A longitudinal study of renal prognosis in patients with IgAN was conducted to search retrospectively for a genotype-phenotype association between RAS polymorphisms and end-stage renal failure (ESRF). A classification based on serum creatinine (S cr ) and 24-h proteinuria (24-P) measured at the time of renal biopsy was used to estimate the risk of ESRF in IgAN: stage 1 (S cr 150 mol/l and 24-P 1 g), stage 2 (S cr 150 mol/l and 24-P 1gorS cr 150 mol/l and 24-P 1 g), stage 3 (S cr 150 mol/l and 24-P 1 g). Deletion/insertion polymorphism (D/I) of the angiotensin I converting enzyme gene, M235T polymorphism (T/M) of the angiotensinogen gene and A1166C polymorphism (C/A) ofthe angiotensin II type 1 receptor gene were determined in 274 Caucasian men with biopsy-proven IgAN (n 86, 112, and 76 in stages 1, 2, and 3, respectively). Mean global follow-up was 6 5 yr after renal biopsy. For stages 1, 2, and 3, ESRF developed in 7 (8.1%), 39 (34.8%), and 49 (64.4%) cases (P ), , 5.4 4, and 2 2 yr, respectively, after renal biopsy (P 0.001). The distributions of the three genotypes into the three stages were similar. Different distributions were observed when patients were grouped by stage and genotype: ID DD: 72% in stage 1 versus 84.6% in stages 2 3 (P 0.02; 0.14); MT TT: 66.2% in stages 1 2 versus 78.9% in stage 3 (P 0.04; 0.09); and AA AC: 89.9% in stages 1 2 versus 97.4% in stage 3 (P 0.04; 0.1). However, with the use of the Cox proportional hazard model, none of the three genotypes was found to have predictive value for renal survival. Compared with S cr and 24-P, genotypes DD, TT, and AA are unlikely to serve as clinically useful predictors of ESRF in IgAN. The renin-angiotensin system (RAS) is one of the main pathophysiologic factors in nephropathies with proteinuria (1). Angiotensin I converting enzyme (ACE) and angiotensinogen (Agt) gene polymorphisms are expressed phenotypically by significant variations in serum levels (2,3), but the prognostic significance remains to be fully defined (2 5). Four of the five clinical studies on IgA nephropathy (IgAN) in adults with an ACE gene deletion/insertion (D/I) reported that genotype DD is a poor prognosis factor (6 10). The two studies (11,12) on subjects with Agt gene M235T (M/T) polymorphism and angiotensin II type 1 receptor (ATR) gene A1166C (C/A) polymorphism in addition to ACE polymorphism were in disagreement. In one report (11), it was found that the DD genotype had an impact on prognosis, whereas in the other (12) only the TT Received January 11, Accepted April 13, Correspondence to Dr. Luc Frimat, Service de Néphrologie, CHU de Nancy, Hôpitaux de Brabois, Vandoeuvre-les-Nancy, France. Phone: ; Fax: ; l.frimat@chu-nancy.fr / Journal of the American Society of Nephrology Copyright 2000 by the American Society of Nephrology genotype had an influence. These contradictory results could be explained by different sample sizes (48 to 204 patients), variable proportions of severe forms, unrecognized stratification of the populations, different end points, and interference of renin-angiotensin inhibitory drugs. Such genetic studies must also meet methodologic prerequisites: Hardy-Weinberg equilibrium and sufficient statistical power (2,3,10,12,13). In a multicentric prospective cohort study on IgAN with a mean 5.6-yr follow-up after renal biopsy (14), we demonstrated that serum creatinine and 24-h proteinuria (24-P) at the time of renal biopsy together with gender are three independent variables that are predictive of end-stage renal failure (ESRF). Serum creatinine and 24-P are two commonly described markers of prognosis (15,16). It has also been recently demonstrated that renal disease progresses more slowly to ESRF in women than in men (17). In previous work (14), we established a prognosis classification of ESRF for men with IgAN: stage 1 (serum creatinine 150 mol/l and 24-P 1 g), stage 2 (serum creatinine 150 mol/l and 24-P 1 g or serum creatinine 150 mol/l and 24-P 1 g), stage 3 (serum creatinine 150 mol/l and 24-P 1 g). Seven years after renal biopsy, ESRF-free survival was 98.5% for stage 1, 86.6%

2 J Am Soc Nephrol 11: , 2000 Renin-Angiotensin System Gene in IgA Nephropathy 2063 for stage 2, and 21.3% for stage 3 (P 0.001). In the present study, we examined the hypothesis that ACE and/or Agt and/or ATR gene polymorphisms correlate with phenotypic distribution in these three prognostic stages. Materials and Methods Patients IgAN was defined as glomerulonephritis with predominantly IgA deposits in the mesangium of all glomeruli. Caucasian patients whose renal biopsy performed after 1968 in 17 nephrology centers (see Acknowledgments) evidenced IgAN were contacted between December 1998 and June 1999 for participation in this study. Women; patients with Henoch-Schönlein purpura, lupus, cirrhosis, or systemic disease; those younger than 10 yr at the time of biopsy; and Black and Asian patients were not included. Patients who were taking drugs that interfere with 24-P measurements (ACE inhibitors [ACEi], angiotensin II receptor antagonists [AT 1 blockers]) were not included. To test the Hardy-Weinberg equilibrium, genotypic distributions were compared with those in a control group composed of 960 Caucasian men in the Stanislas cohort followed at the Nancy region preventive medicine center (18). In this group, the genotypic distributions were II/20.5%, ID/49.5%, and DD/30% for ACE and AA/49%, AC/42%, CC/9% for ATR. Clinical Variables We recorded age, history of gross hematuria, serum creatinine, 24-P, systolic and diastolic BP, and antihypertensive therapy at the time of the histologic diagnosis. For patients who progressed to ESRF, the date of onset of substitution therapy was noted. For patients who did not develop ESRF, serum creatinine, 24-P, systolic and diastolic BP, and antihypertensive therapy at the time the genotype was determined were recorded. The duration of the follow-up since the renal biopsy was determined. To take into account changes in indications for renal biopsy from 1992, biopsies were obtained only in patients with severe disease a variable indicating the period during which the biopsy was performed was included in the multivariate analyses. Definition of Outcome This study was longitudinal and retrospective. ESRF was the outcome criterion. The risk of ESRF was estimated from serum creatinine and 24-P at the time of the renal biopsy. At the time of the genotyping, patients were retrospectively classed into one of the three stages described above. Genetic Study Informed consent was obtained from each person. Nuclear DNA was extracted with the Nucleon Bacc3 system resin (Amersham, England) which gives good quality high-molecular-weight DNA from leukocyte extracts of 15ml peripheral venous blood samples. The D/I study was performed on a 287 base-pair (bp) fragment of the ACE gene intron 16 using in vitro elective amplification in the presence of Taq polymerase, genomic DNA, and specific primers described by Hunley et al. (11). Because of the preferential amplification of the D allele compared with the larger I allele, ID heterozygotes can be confused with DD homozygotes. To detect these falsepositive DD, all of the samples with a DD phenotype were checked with PCR using a pair of primers specific for the insertion sequence whose amplification product is expected at 335 bp. A search for Agt gene polymorphism was performed with the PCR method described by Russ et al. (19) using specific primers that produce a 165-bp fragment including a nonsense point mutation in exon 2 causing methionine (M) substitution for threonine (T) at position 235 (M235T). The point mutation with adenosine (A) substitution for cytosine (C) in nucleotide 1166 in the untranslated 3 part of the ATR gene creates a restriction site for the Dde I enzyme. PCR amplification with specific primers produces a 546-bp fragment. Digestion with Dde I generates two 435- and 111-bp fragments uniquely in the presence of the C allele (20). For each of these three polymorphisms, control DNA samples from subjects with known genotype were systematically included in each experiment as were samples totally devoid of DNA. Statistical Analyses Deviation from Hardy-Weinberg equilibrium was assessed by goodness of fit between the observed and expected number of each genotype with 2 test. Qualitative variables for the three stages were compared with 2 test and continuous variables with ANOVA. Kruskal-Wallis analysis was performed for variables with a nonnormal distribution. The prognostic values of each of the three genotypes (ACE, Agt, ATR) were established in two ways. First, we compared the stage distributions and the genotype distributions using 2 test and the concordance of these distributions with test. Second, to assess the direct prognostic impact of the genotypes on renal survival and to search gene variable or gene gene interactions, the Cox model was used for univariate then multivariate analyses. BMDP statistical software (Los Angeles, CA) was used for all statistical analysis. Sample Size The number of subjects required for statistical significance was calculated with Solo Power Analysis Software. Under the hypothesis that the genes are independent, search for distribution concordance was done with test with an accepted risk of 5% for a two-way test and 80% power. Accepting a 90% concordance and 0.8, the necessary sample size was 270 patients. The second approximation estimated the number of subjects necessary to evidence a relative risk corresponding to different renal survival rates for the three stages. For the smallest estimated relative risk with 80% power and risk of 2% for a two-way test, the number of subjects required was an estimated 500. Finally, we decided to set our inclusion goal at 300 subjects (100 per stage). Results The study group (IGA nephropathy and the genetic polymorphism in the Renin-Angiotensin System [IGARAS] Study; see Acknowledgments for participating centers and laboratories) included 274 male Caucasians with IgAN proved on biopsies performed from 1968 through Mean age at the time of the renal biopsy was yr (range, 12.4 to 86 yr). Mean follow-up since biopsy was 6 5 yr (range, 0.1 to 29 yr). Ninety-five patients (34.6%) had progressed to ESRF. The clinical features of the patients by prognostic stage are given in Table 1. The percentage of patients with ESRF and the time interval from biopsy to ESRF were significantly different for the three stages (P 0.001). There was no departure from the Hardy-Weinberg equilibrium for genotypic distributions for ACE (P 0.72) and ATR (P 0.86). As shown in Table 2, the distributions of the three genotypes

3 2064 Journal of the American Society of Nephrology J Am Soc Nephrol 11: , 2000 Table 1. Clinical and biological variables a Variables Stage P Value Variables at histologic diagnosis number of patients [n (%)] 86 (31.4) 112 (40.9) 76 (27.7) age (yr) h proteinuria (g) serum creatinine ( mol/l) systolic BP (mmhg) diastolic BP (mmhg) antihypertensive drug (number) Variables at end of follow-up ESRF in each stage [n (%)] 7 (8.1) 39 (34.8) 49 (64.4) time between biopsy and ESRF (yr) Variables at last medical evaluation for patients without ESRF number of patients (n) follow-up after biopsy (yr) age (yr) h proteinuria (g) serum creatinine ( mol/l) systolic BP (mmhg) diastolic BP (mmhg) antihypertensive drug (number) ACEi (%) AT 1 blockers (%) a Results are given as mean SD. BP, blood pressure; ESRF, end-stage renal failure; ACEi, angiotensin I converting enzyme inhibitors; AT 1 blockers, angiotensin II receptor antagonists. into the three prognostic stages were similar. To achieve more statistical power, 2 tests were run again after grouping stages and genotypes. Significant differences in distributions (P 0.05) were found for the groups presented in Table 3. Compared with stages 2 and 3 grouped together, the proportion of genotype II in stage 1 was higher than that of genotypes ID and DD together. The proportion of genotypes MM and CC was higher than that of genotypes MT and TT together and AC and AA together in stages 1 and 2 taken together compared with stage 3. However, for all of these comparisons, the observer agreement was slight or poor, i.e., less than 20%, meaning that if there was any resemblance between stage and genotype, it was weak. Using Cox proportional hazards model, univariate analysis showed that ACE, Agt, and ATR did not have a significant prognostic impact. Stage alone remained as an independent variable after multivariate analysis using the same prognostic values as in our preceding study (age at biopsy, stage, systolic BP, gross hematuria, and the period variable). Compared with stage 1, the relative risk of ESRF was 5.5 (95% confidence interval [CI], 3.8 to 7.8) in stage 2 patients and 30.2 (95% CI, 14.4 to 60.8) in stage 3 patients. To assess directly the prognostic impact of genotypes on renal survival in this multivariate analysis, the stage variable was successively replaced by the ACE, Agt, and ATR variables. None of these three genotypes had any prognostic impact. In passing, an interaction between systolic BP and ATR was found, i.e., AA genotype patients with hypertension had a higher relative risk of ESRF than CC genotype patients with hypertension (P 0.038). No gene gene interaction was found. The IGARAS study, designed to confirm or refute previous findings, yielded a highly significant negative result. Indeed, the power of the study to detect an underlying difference ( error analysis) was greater than 80%. Discussion Since the mid 1990s, a considerable amount of work has been devoted to RAS gene polymorphisms (2 5) as part of investigations into factors that predispose to multifactorial cardiovascular and renal diseases (21). To obtain an accurate estimate of the clinical impact of genotype in these diseases, pathophysiologic mechanisms, detailed description of all of the

4 J Am Soc Nephrol 11: , 2000 Renin-Angiotensin System Gene in IgA Nephropathy 2065 Table 2. Genotype frequency in each stage a Stage ACE (%) II ID DD ; P 0.11 Agt (%) MM MT TT ; P 0.16 ATR (%) AA AC CC ; P 0.12 a ACE, angiotensin I converting enzyme; I/D, insertion/deletion; Agt, angiotensinogen; M/T, M235T; ATR, angiotensin II type 1 receptor; C/A, A1166C. disease phenotypes, and environmental factors must be taken into account simultaneously (21,22). IgAN is one such multifactorial disease with a largely unexplained pathogenesis. The bias as a result of global assessment of glomerular diseases that result from different pathogenic mechanisms (23) was avoided in our study and in others (6 12) because only patients with IgAN were included. Epidemiology and experimental studies have shown that the renal disease progresses more slowly to ESRF in women than in men (4,14,17). To avoid the potential confounding effect of gender on prognosis, the IGARAS study, unlike previous studies (6 12), included only men. Men in the three prognosis stages have very different relative risks for ESRF. Unrecognized stratification of the populations that possibly occurred in certain studies was avoided. Among the seven studies on the prognostic impact of gene polymorphism in IgAN, only one used ESRF as the end point (8). The others used slope of serum creatinine (6), loss of clearance of creatinine (7,9,12), doubling serum creatinine or ESRF (10), or a cutoff point for serum creatinine (11). In IgAN, the increase of serum creatinine and the decrease of clearance of creatinine are known to fluctuate with time. Estimations of prognostic impact are subsequently less precise when these variables are used as the end point (1,14,16,24). In the IGARAS study, all patients who were taking a drug that interfered with 24-P (ACEi or AT 1 blockers) were excluded. In addition, the study was based on a classification established at a period (1987 through 1996) when slower progression of chronic renal failure was not yet an indication for ACEi. Finally, it is reasonable to assume homogeneous salt intake in our Caucasian population from eastern and northern France. The environmental factors involved in IgAN were thus accounted for (21). Before the IGARAS study, we estimated the number of subjects required to demonstrate a significant difference, taking into account the classification (14) and the distribution of the genotypes in our control population (18). This approach led us to include a large group of 274 patients to avoid the biases generated by small sample sizes in certain studies. In addition, the IGARAS study is in agreement with the Hardy-Weinberg equilibrium theoretical model. In 1997, Pei et al. (12) observed that only three (6,11,12) of the six studies devoted to D/I polymorphism in patients with progressing chronic renal failure were in agreement with the Hardy-Weinberg law. In the other studies, this bias could have caused less accurate assessment of prognostic markers (13). The results of IGARAS study show that the distributions of the three genotypes for the three markers into the three prognosis stages were similar (Table 2). After grouping genotypes and stages, the proportions of genotypes II, MM, and CC were higher in stages with better prognosis (Table 3). However, the very small observer agreement is not in favor of a cause-andeffect relationship. The multivariate analysis demonstrated clearly that compared with proteinuria, high BP, and serum creatinine, genotypes have no independent prognostic value. Finally, our study suggests that in glomerular disease, the respective contributions of each of the three RAS gene polymorphisms to predicting progression to irreversible renal parenchymal damage are weak. These results confirm those re- Table 3. Genotype frequency after grouping stage and genotypes Genotype Stage 1 1 and 2 2 and 3 3 ACE (%) II ID and DD ; P 0.02; 0.14 Agt (%) MM MT and TT ; P 0.04; 0.09 ATR (%) AA and AC CC ; P 0.04; 0.1

5 2066 Journal of the American Society of Nephrology J Am Soc Nephrol 11: , 2000 ported by Schmidt et al. (8), who used the same end point and the largest study population. The earlier studies on ACE polymorphism reported seemingly contradictory results (6 12). Indeed, a meta-analysis of five studies (6 9,11) did not evidence any prognostic impact for genotypes ID and DD compared with genotype II (odds ratio and corresponding 95% CI, 0.7 [0.5 to 0.9], 1.2 [0.8 to 1.8]) (2). For Agt polymorphism, our study did not confirm the negative prognostic impact of genotype TT reported by Pei et al. (12). These differences could be explained by variations in their designs. Like two previous studies (11,12), our study did not evidence any impact of genotype AA on prognosis. Unlike ACE and Agt polymorphism, ATR polymorphism is considered to be nonfunctional (4). The weak synergetic effect of genotype AA and high BP on renal prognosis evidenced here was previously suspected (5). In conclusion, the IGARAS study, designed to quantify the prognostic impact of RAS gene polymorphism in IgAN, has yielded negative results with powerful statistical significance. The relationships between genotype and phenotype explain, in part, the variance of serum ACE and Agt levels. This observation highlights only a restricted part of the overall participation of the RAS in the pathophysiology of chronic nephropathies. Furthermore, on the basis of a recent study (25), it seems that variations in ACE gene are much more complex than envisioned. Thus, although our results tend to exclude determined genetic differences of the RAS as factors that have an important impact on the progression of IgAN to ESRF, they do not exclude a causal role for the RAS in the pathogenesis of proteinuric nephropathies (1). Acknowledgments This study was funded by a Clinical Research Program grant from the University Hospital of Nancy, France. The IGARAS Study Group consists of the following investigators [number of recruited patients]: CHU de Nancy [78]: clinical coordinating center (M. Kessler, L. Frimat, J.L. André, B. Aymard [Centre de pathologie, Metz], M. Bellou, T. Cao Huu, J. Champigneulles, P. Clavel, D. Hestin, M.J. Krier, M.N. Maghakian, E. Renoult), genetic section (P. Jonveaux, C. Philippe, C. Demmerle), biostatistics section (S. Briançon, F. Guillemin); CHU de Caen [24] (B. Hurault de Ligny, S. Fradin); Hôpital Manchester de Charleville-Mézières [21] (J.J. Dion); Hôpital Pasteur de Colmar [10] (B. Faller, S. Chiron); Hôpital Pasteur de Dôle [5] (J. Guillaumie); CHU de Lille [24] (M. Hazzan, C. Noël); Hôpital Bon Secours de Metz [2] (P. Mirgaine, H. Terrasse); Hôpital Saint André de Metz [7] (E. Azoulay, M. Galy, P. Gautier); Polyclinique de Gentilly et d Essey [4] (J.M. Bertheau, J.C. Valdenaire); CHU de Reims [18] (J. Chanard, P. Birembaut, F. Lacour, H. Maheut, J.P. Melin); Clinique Bethesda de Strasbourg [2] (J.F. Marichal); Hôpital Bel Air de Thionville [8] (P.L. Caraman, F. Comte); Hôpital de Troyes [22] (T. Milcent, R. Montagnac, F. Schillinger); Hôpital de Valenciennes [16] (V. Lemaitre, P. Vanhille); Hôpital Saint Nicolas de Verdun [9] (P. Bindi, B. Gilson); Hôpital Paul Morel de Vesoul [12] (B. Guyon, L. Lamriben, J.P. Ory); Hôpital de Vittel [12] (J.Y. Hesse, E Prenat, H. Sekhri). References 1. Remuzzi G, Bertani T: Pathophysiology of progressive nephropathies. N Engl J Med 339: , Staessen JA, Wang JG, Ginocchio G, Petrov V, Saavedra AP, Soubrier F, Vlietinck R, Fagard R: The deletion/insertion polymorphism of the angiotensin converting enzyme gene and cardiovascular-renal risk. J Hypertens 15: , Staessen JA, Kuznetsova T, Wang JG, Emelianov D, Vlietinck R, Fagard R: M235T angiotensinogen gene polymorphism and cardiovascular renal risk. 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6 J Am Soc Nephrol 11: , 2000 Renin-Angiotensin System Gene in IgA Nephropathy Siest G, Visvikis S, Herbeth B, Gueguen R, Vincent-Viry M, Sass C, Beaud B, Lecomte E, Steinmetz J, Locuty J, Chevrier P: Objectives, design and recruitment of a familial and longitudinal cohort for studying gene-environment interactions in the field of cardiovascular risk: The Stanislas cohort. Clin Chem Lab Med 36: 35 42, Russ AP, Maerz W, Ruzicka V, Stein U, Gross W: Rapid detection of the hypertension-associated Met 235 3Thr allele of the human angiotensinogen gene. Hum Mol Genet 2: , Doria A, Ji L, Warram JH, Krolewski A: DdeI polymorphism in the AGTR1 gene. Hum Mol Genet 3: 1444, Cambien F: Genetic epidemiology and frequent multifactorial diseases. Rev Epidemiol Sante Publique 44: , Lander ES, Schork NJ: Genetic dissection of complex traits. Science 265: , Van Essen GG, Rensma PL, de Zeeuw D, Sluiter WJ, Scheffer H, Apperloo AJ, de Jong PE: Association between angiotensinconverting-enzyme gene polymorphism and failure to renoprotective therapy. Lancet 347: 94 95, Levey AS, Gassman JJ, Hall PM, Walker WG: Assessing the progression of renal disease in clinical studies: Effects of duration of follow-up and regression to the mean. J Am Soc Nephrol 1: , Rieder MJ, Taylor SL, Clarck AG, Nickerson DA: Sequence variation in the human angiotensin converting enzyme. Nat Genet 22:59 62, 1999 Access to UpToDate on-line is available for additional clinical information at