Regression of left ventricular hypertrophy by lisinopril after renal transplantation: Role of ACE gene polymorphism

Kidney International, Vol. 58 (2000), pp. 889–897

Regression of left ventricular hypertrophy by lisinopril after renal transplantation: Role of ACE gene polymorphism DOMINGO HERNA´NDEZ, JUAN LACALZADA, EDUARDO SALIDO, JOSE´ LINARES, ANTONIO BARRAGA´N, VI´CTOR LORENZO, LUZ HIGUERAS, BASILIO MARTI´N, AURELIO RODRI´GUEZ, IGNACIO LAYNEZ, JOSE´ M. GONZA´LEZ-POSADA, and ARMANDO TORRES Nephrology and Cardiology Services, and Unidad de Investigacio´n, Hospital Universitario de Canarias, Tenerife, Spain

reduction was more evident in DD patients (placebo DD, 8.4 ⫾ 4.1% vs. lisinopril DD, ⫺7.2 ⫾ 5.3, P ⬍ 0.05), and a trend was observed in patients with other genotypes (placebo ID/II, 2.8 ⫾ 5.4% vs. lisinopril ID/II, ⫺11.4 ⫾ 5%, P ⫽ 0.33). Conclusions. Lisinopril decreases LVM in renal transplant patients with hypertension and LVH, and the ACE gene polymorphism may predict the beneficial effect of this therapy. This finding may be important in targeting prophylactic interventions in this population.

Regression of left ventricular hypertrophy by lisinopril after renal transplantation: Role of ACE gene polymorphism. Background. Cardiac complications are the main cause of death in renal transplantation (RT), and left ventricular hypertrophy (LVH) may play an important role in these patients. The unfavorable genotype of the angiotensin-converting enzyme (ACE) gene has been associated with cardiovascular disease, including LVH. ACE inhibitors (ACEIs) reduce LVH, but little is known about the effects of ACEIs on LVH in RT patients with different insertion/deletion (I/D) genotypes of the ACE gene. Methods. We prospectively studied 57 stable nondiabetic RT patients with hypertension and echocardiographic LVH as well as a functional graft for 69.5 ⫾ 5.6 months. Patients randomly received either lisinopril 10 mg/day (group A, N ⫽ 29; 5 were excluded due to reversible acute renal failure) or placebo (group B, N ⫽ 28) for 12 months. Echocardiography (M-mode, 2-B, and color flow Doppler) was performed at baseline and 6 and 12 months later by the same examiner without previous knowledge of the genetic typing. The ACE genotype (I or D alleles) was ascertained by polymerase chain reaction (PCR; group A, DD ⫽ 10 and ID/II ⫽ 14; group B, DD ⫽ 15 and ID/II ⫽ 13). Results. All patients maintained a good renal function (serum creatinine ⬍2.5 mg/dL) during the follow-up and both groups received a similar proportion of antihypertensive drugs (␤-blockers 83 vs. 79%; Ca antagonists 66 vs. 68%; ␣1-adrenoreceptor antagonists 50 vs. 67%) during the study. As expected, mean arterial blood pressure and hemoglobin levels showed a higher percentage reduction in group A versus group B (⫺4 ⫾ 2.8 vs. 2.1 ⫾ 2.6%, P ⫽ 0.07, and ⫺11.5 ⫾ 1.5 vs. ⫺0.5 ⫾ 2.3%, P ⬍ 0.01, respectively). Group A patients showed a significantly higher decrement in LV mass index (LVMI) than group B at the end of follow-up, after adjusting for age, baseline LVMI, time after grafting and changes in systolic blood pressure, renal function, and hemoglobin levels (group A, ⫺9.5 ⫾ 3.5% vs. group B, 3 ⫾ 3.2%, P ⬍ 0.05). As a result, 46% of group A and only 7% of group B patients showed a reduction of LVMI ⱖ15% (P ⬍ 0.01). The beneficial effect of lisinopril on LVMI

Cardiac complications are the main cause of death in renal transplant patients, and left ventricular hypertrophy (LVH) is considered a major independent risk factor [1–3]. LVH is common in these patients, and some factors in the process of transplantation, mainly hypertension, have been implicated [4]. In addition, antirejection therapy (corticosteroids and cyclosporine) could be also involved in the development of LVH [5, 6]. Several experimental studies have documented the growth-stimulating and regulating effect of angiotensin II on myocardial cells [7, 8]. Angiotensin-converting enzyme (ACE) is a key enzyme in the production of this substance, and it may therefore participate in the cardiac protein synthesis [9]. Conversely, pharmacological inhibition of the angiotensin II synthesis with ACE inhibitors (ACEIs) reduced LVH in hypertensive patients more effectively than other antihypertensive drugs [10, 11]. However, this effect has not been explored after renal transplantation (RT). An insertion (I)/deletion (D) polymorphism of the ACE gene (ACE/ID) has been associated with cardiovascular diseases [12, 13], including LVH [14–16]. In dialysis patients, DD genotype has been associated with a higher left ventricular mass (LVM) [17]. Recently, we have also observed that the presence of DD genotype is an independent risk factor favoring LVH in renal transplant patients [18]. The beneficial response to ACEIs may be influenced by the ACE/ID polymorphism. Individual differences on the antiproteinuric or renoprotective effect of ACEIs in renal transplant patients have been ob-

Key words: hypertension, cardiovascular disease, acute renal failure, uremia, angiotensin-converting enzyme gene polymorphism. Received for publication June 18, 1999 and in revised form February 11, 2000 Accepted for publication March 14, 2000

 2000 by the International Society of Nephrology

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served [19, 20]. The effect of ACEIs on LVM changes may be also influenced by this polymorphism of the ACE gene [21], but this issue has not been explored in renal transplant patients. The present randomized study was undertaken to assess the long-term effect of an ACEI, lisinopril, on echocardiographic, morphological and functional changes in hypertensive renal transplant patients with LVH, whose ACE genotype was also determined. METHODS The study was performed in a single-dose, single-blind, randomized, placebo-controlled design during 12 months. Patients Study participants were stable nondiabetic, long-term renal transplant patients of both sexes (25 to 70 years old) with arterial hypertension and echocardiographic evidence of LVH. Inclusion criteria were the following: (1) presence of hypertension, defined as a systolic and diastolic blood pressure ⬎150/90 mm Hg, requiring two or more antihypertensive drugs for adequate blood pressure control; (2) no previous treatment with ACE inhibitors or angiotensin II receptor antagonists; (3) stable renal function, with a serum creatinine level ⬍2.5 mg/dL or ⬍221 ␮mol/L for more than six months; (4) presence of LVH, assessed by echocardiographic criteria; (5) absence of clinical features of heart failure or significant valvular disease; (6) absence of renal artery stenosis or chronic allograft nephropathy; (7) no documentation or clinical suspicion of renovascular hypertension; and (8) absence of proteinuria. Exclusion criteria during follow-up were: (1) increment of serum creatinine level by 20% or more during treatment, (2) severe side effects, (3) detection of chronic allograft nephropathy, and (4) pregnancy. Women were advised about physical anticonceptive measures during follow-up. In all patients, immunosuppression consisted of antithymocyte globulin (ATGAM; Upjohn, Kalamazoo, MI, USA) for induction, and prednisone, cyclosporine, and azathioprine for maintenance. Recipients older than 50 years did not receive azathioprine. Episodes of acute rejection were initially treated with three boluses of 500 mg of intravenous methylprednisolone. Resistant episodes were treated with a 10-day course of OKT3 (5 mg/ day). Cyclosporine was adjusted according to the total blood levels [22]. The study was approved by the Ethics Committee of the Hospital Universitario and by the Direccio´n General de Farmacia (Spanish Ministry of Health, Madrid, Spain). This study was conducted according to the Declaration of Helsinki, and each patient gave written informed consent to participate in the study.

Sample size The planned size was 60 patients, which was based on the assumption of a reduction of at least 15% in LVM by ACEIs, and less than 3% in the placebo group. With a two-tailed ␣ ⫽ 0.05, the study of 60 patients was expected to have 80% power to detect an overall LVM reduction of 15%, based on an intention-to-treat analysis. Study objectives The primary objective was to investigate the effect of treatment with lisinopril on the rate of change in LVM in stable hypertensive renal transplant patients with LVH. A secondary objective was to assess the influence of the ACE/ID polymorphism on the cardiac changes induced by lisinopril. In addition, adverse events, renal function, and blood pressure response were also regularly recorded in each group. Study design After screening assessment, all eligible patients entered a one-month single-blind, placebo run-in phase. At the end of this period, patients with capsule compliance exceeding 80% were enrolled in the study. Patients were randomly assigned to receive either 10 mg/day of lisinopril (group A, N ⫽ 29) or placebo (group B, N ⫽ 28) for 12 months. Because lisinopril provides relatively constant plasma concentration of the drug over 24 hours [23], patients were instructed to take the medication at a convenient but consistent time of the day to ensure compliance. All of the tablets had identical appearance and were provided by Vita S.A. (Barcelona, Spain). Clinical and biochemical data were measured at the randomization visit and every three months thereafter. Initially, evaluation of renal function was performed twice weekly. Lisinopril was discontinued if plasma creatinine had increased by ⱖ20% during this period. This cut-off value was based on previous studies in which a minimum increase in plasma creatinine during ACEI treatment of ⱖ20% was considered significant [24]. On the other hand, if plasma creatinine had not changed or increased less than 20%, lisinopril was continued during follow-up. The goal of antihypertensive therapy was to obtain a blood pressure ⱕ145/90 mm Hg in both groups during the study. Thus, antihypertensive agents, other than ACEIs or angiotensin II receptor antagonists, were adjusted to achieve this blood pressure control during follow-up as in standard clinical practice. Echocardiographic parameters were measured at baseline and at 6 and 12 months later. All patients were asked about adverse events at each visit. Serious adverse events were defined as withdrawal for any serious medical reason, death, any major morbid event or hospital admission for any reason, and any ab-

Herna´ndez et al.: Lisinopril after renal transplantation

normal laboratory value associated with signs or symptoms or requiring treatment. Withdrawal from treatment could result from a serious adverse event or from the patient’s wishes. Laboratory measurements Serum creatinine and other biochemical parameters (uric acid, glucose, and complete blood count) were measured by means of a computerized autoanalyzer (Hitachi 717; Boehringer Mannheim, Mannheim, Germany). Total blood cyclosporine levels were quantitated by fluorescence polarization immunoassay (FPIA) using a monoclonal antibody (Abbot, IL, USA). Echocardiographic technique and data collection M-mode, two-dimensional, and color-flow Doppler echocardiograms were performed by the same examiner (J.L.) without previous knowledge of the genetic typing, clinical characteristics, and study group, both at baseline and after follow-up. Each echocardiographic tracing was coded and interpreted at random by a second experienced observer (A.B.), who was also unaware of the subjects’ identity, clinical characteristics, and treatment group. In our laboratory, interobserver variability for these measurements in 10 repeated studies averaged ⬍10% [18]. Echocardiograms were obtained with the patient in the left decubitus position with 30⬚ head inclination, using an ultrasonoscope (Aloka SSZ-203) with either a 2.5 or a 3.5 MHz transducer. The left ventricular echocardiogram was recorded following a standard protocol at or just below the tips of the mitral valve leaflets, with the transducer applied to the third or fourth intercostal space. Measurements of cavities and cardiac walls were undertaken following the recommendations of the American Society for Echocardiography [25]. LVM was determined according to the method of Devereux and Reichek [26] and indexed to body surface area to yield the LVM index (LVMI). The cut-off level defining LVH was a LVMI ⬎ 143 g/m2 in men and LVMI ⬎ 102 g/m2 in women [27]. Left ventricular systolic function was assessed by the ejection fraction, which was calculated as LVEDV-LVESV/ LVEDV where LVEDV and LVESV are end-diastolic and end-systolic volumes both calculated by the formula of Teichholz et al [28]. The Doppler echocardiography was performed immediately after the M-mode examination. Transmitral flow velocity signals were recorded simultaneously with an electrocardiogram and a phonocardiogram. The following measurements were obtained: maximal early (peak E) and late (peak A) diastolic flow velocities in centimeters per second, deceleration time of flow velocity in early diastole (DT), and left ventricular isovolumic relaxation time (LVIRT) in milliseconds. In addition, the ratio of peak flow velocity in early diastole to peak flow velocity during atrial systole (E/A) was calculated.

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The mean of measurements from three to five consecutive cardiac cycles was determined for each of these indices. Genomic typing of the ACE deletion/ insertion polymorphism The previously described D and I alleles of the ACE gene were ascertained by polymerase chain reaction (PCR) amplification of a fragment of intron 16 of the ACE gene [29]. DNA was purified from 3 mL of blood by proteinase K digestion, phenol extraction, and ethanol precipitation, following standard protocols [30]. Approximately 0.1 ␮g DNA was amplified with primers hace3s, 5⬘-GCCCTGC AGGTGTCTGCAGCATGT-3⬘, and hace3as, 5⬘-GGA TGGCTCTCCCCGCCTTGTCTC-3⬘, which yield PCR products of 319 bp and 597 bp for the D and I alleles, respectively. PCR conditions included 10 mmol/L TrisHCl, pH 9.0, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.1% Triton X-100, 0.1 mmol/L dNTPs, 0.5 ␮mol/L each primer, and 1 U Taq DNA polymerase. A Perkin-Elmer 480 thermal cycler was used to carry out 30 amplification cycles with the following temperature profile: 94⬚C (1 min), 62⬚C (1 min), and 72⬚C (1 min). Ten microliters of the amplification product were resolved by electrophoresis in 5% polyacrylamide gels and visualized under ultraviolet light after staining with 0.5 ␮g/mL ethidium bromide. All samples yielding exclusive amplification of the D allele (and therefore potentially typed as DD) were subjected to a second independent amplification, as described by Lindpaintner et al [29]. Briefly, samples were amplified under the same buffer conditions as described previously in this article, except for the use of primers specific for the sequence inserted in intron 16 of the I allele, and a temperature profile of 94⬚C, 67⬚C, and 72⬚C for one minute each. The primer sequences are hace5a, 5⬘-TGGGACC ACAGCGCCCGCCACTAC-3⬘, and hace5c, 5⬘-TCGC CAGCCCTCCCATGCCCATAA-3⬘, which result in an amplification product of 335 bp only in the presence of an I allele. Using the same procedure, we also estimated the allele frequencies for the I/D polymorphism at the ACE locus among 246 DNA samples from healthy individuals and among 248 unselected DNA samples from renal transplant patients. Statistical analysis Friedman two-way analysis of variance (ANOVA) by rank test was used to compare repeated echocardiographic measurements during the study. Intragroup pair-wise comparisons were performed by the Wilcoxon signedrank test with adjusted degrees of freedom. The general factorial analysis of variance (ANCOVA) was used to measure the treatment effect on the rate of change of different biological variables, after adjusting for other potential confounding covariables. For univariate com-

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parison of numerical variables, U-Mann–Whitney was used. All categorical variables were analyzed using chisquare test or Fisher’s exact probability test (two tailed) as appropriate. Results are expressed as mean ⫾ SE. A P value less than 0.05 was considered significant. All computations were made using the SPSS 7.5 statistical package for Windows威 (Chicago, IL, USA). Genetic statistics were performed with Genepop v.3.1. RESULTS Of the 57 patients who were recruited, 5 patients of the lisinopril group initially withdrew from the study for reversible acute renal failure (increment of level serum creatinine ⱖ20% during the first week). Thus, 24 patients were assigned to receive lisinopril and 28 to receive placebo, and all of them completed the study. No drugrelated side effects were observed in this cohort completing the study, and no patients suffered cardiac failure or angina pectoris during follow-up. Table 1 summarizes clinical, immunosuppression, and renal function data by treatment groups at baseline and at 6 and 12 months later. A longer time after grafting was observed in patients receiving placebo. Both groups received a similar proportion of other antihypertensive drugs during the study. Moreover, systolic, diastolic, and mean arterial pressure were not different at baseline. However, there was a trend to lower values in mean arterial pressure at the end of follow-up in the lisinopril group (Table 1). As expected, a greater reduction of hemoglobin levels was observed in the lisinopril group. Other clinical data such as renal function, antirejection therapy, and body mass index were similar in both groups. Morphological echocardiographic data from both groups are summarized in Table 2. Patients receiving lisinopril showed a significant decrease in LVMI at end of followup, as compared with the placebo group (unadjusted data), and the highest specific reduction was, on average, for the posterior wall (Table 2). However, a trend toward higher baseline LVMI was observed in the lisinopril group. Thus, a general factorial analysis was applied using LVMI as the dependent variable, and treatment (placebo or lisinopril) as factor, while adjusting for age, baseline LVMI, time after grafting, and changes in systolic blood pressure, renal function, and hemoglobin levels. Interestingly, the mean adjusted decrease in LVMI was again significantly greater in the lisinopril group. As a result, 46% of patients receiving lisinopril and only 7% of patients on placebo showed regression of LV mass ⱖ15% (P ⬍ 0.01; Table 2). Other echocardiographic parameters were similar in both groups during the study. Table 3 shows Doppler echocardiographic data during follow-up. Overall diastolic function was similar in both groups during the study.

Table 1. Demographic, clinical and immunosuppression data at baseline, and 6 and 12 months after treatment in both groups

Age years Sex M/F Time after renal transplant months Antihypertensive drugs % ␤-blockers Ca antagonists ␣1-adrenoreceptor antagonists Systolic blood pressure mm Hg Baseline 6 months 12 months Diastolic blood pressure mm Hg Baseline 6 months 12 months Mean arterial pressure mm Hg Baseline 6 months 12 months Mean arterial pressure change % 12 months after treatment Cumulative steroids g CsA levels ng/mL Baseline 6 months 12 months Body mass index kg/m2 Baseline 6 months 12 months Serum creatinine mg/dL Baseline 6 months 12 months Hemoglobin levels g/dL Baseline 6 months 12 months Hemoglobin level change % ACE genotype number of patients

Group A: Lisinopril N ⫽ 24

Group B: Placebo N ⫽ 28

46.9 ⫾ 2.6 16/8

50.2 ⫾ 2.5 18/10

56.5 ⫾ 3.8a

77 ⫾ 5.6

83% 66% 50%

79% 68% 67%

147.6 ⫾ 4.3 145.5 ⫾ 4 140.6 ⫾ 3.9b

147.9 ⫾ 2.8 153.6 ⫾ 2.9 150.7 ⫾ 3.7

90.6 ⫾ 3.3 88.7 ⫾ 2.9 85.4 ⫾ 2.2

89.7 ⫾ 2.5 89.6 ⫾ 1.8 90.7 ⫾ 2.3

109.6 ⫾ 3.3 107.7 ⫾ 2.9 103.8 ⫾ 2.5b

109.1 ⫾ 2.1 110.9 ⫾ 1.6 110.7 ⫾ 2.4

⫺4 ⫾ 2.8c 19.7 ⫾ 1.1

2.1 ⫾ 2.6 21.6 ⫾ 1.4

215 ⫾ 10.3 222.6 ⫾ 9.6 195 ⫾ 7.4

235.8 ⫾ 10.3 215.2 ⫾ 8.9 205.7 ⫾ 7.4

28.4 ⫾ 1.07 28.1 ⫾ 1 28 ⫾ 1

28.4 ⫾ 0.6 28.8 ⫾ 0.7 28.6 ⫾ 0.7

1.48 ⫾ 0.07 1.49 ⫾ 0.07 1.5 ⫾ 0.06

1.5 ⫾ 0.08 1.34 ⫾ 0.07 1.4 ⫾ 0.08

14.1 ⫾ 0.3 12.8 ⫾ 0.27a 12.4 ⫾ 0.32a ⫺11.5 ⫾ 1.5a 10 DD, 13 ID, 1 II

13.8 ⫾ 0.3 14.2 ⫾ 0.3 13.7 ⫾ 0.3 ⫺0.5 ⫾ 2.3 15 DD, 12 ID, 1 II

Conversion factors to SI units: Creatinine 88.4 (␮mol/L). a P ⬍ 0.01, bP ⫽ 0.05, cP ⫽ 0.07 vs. Placebo group

All samples typed as DD with the first assay (primer hace3s and hace3as) were confirmed to lack the I allele by using the second assay (primers hace5a and hace5c), and no disagreement was found between both assays. The distribution of the DD, ID, and II genotypes in our patients was 48.1% (N ⫽ 25), 48.1% (N ⫽ 25), and 3.8% (N ⫽ 2), respectively, with estimated allele frequencies of 72.1% (D) and 27.9% (I). For the 246 samples from healthy individuals, the following frequencies were obtained: 37.8% DD, 49.2% ID, 13% II, with estimated allele frequencies of 62.4% (D) and 37.6% (I). Among the 248 unselected transplant recipients, 39.5% were DD, 50.4% ID, 10.1% II, with estimated allele frequencies of 64.7% (D) and 35.3% (I). Genotype frequencies

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Herna´ndez et al.: Lisinopril after renal transplantation Table 2. Echocardiographic parameters at baseline, and 6 and 12 months after treatment in both groups

LVESD mm Baseline 6 months 12 months LVEDD mm Baseline 6 months 12 months LAD mm Baseline 6 months 12 months LVEF % Baseline 6 months 12 months IVST mm Baseline 6 months 12 months PWT mm Baseline 6 months 12 months LVMI g/m2 Baseline 6 months 12 months LVMI change % 6 months 12 months unadjusted adjusted Patients with regression of LV mass ⱖ15% at 12 months %

Table 3. Doppler echocardiographic measurements at baseline, and at 6 and 12 months after treatment

Group A: Lisinopril N ⫽ 24

Group B: Placebo N ⫽ 28

28 ⫾ 0.8 29.3 ⫾ 0.8 28.1 ⫾ 1.2

29 ⫾ 1.4 27.9 ⫾ 1.1 27.4 ⫾ 1.2

50.1 ⫾ 1 52 ⫾ 0.9 51 ⫾ 0.9

48.7 ⫾ 1.3 50.3 ⫾ 1.2 49.8 ⫾ 1.3

39.7 ⫾ 1.2 41 ⫾ 1.3 39 ⫾ 1.6

39.8 ⫾ 1 41.1 ⫾ 0.9 39.8 ⫾ 0.9

72 ⫾ 1.5 70.8 ⫾ 1.4 70.8 ⫾ 2

70.5 ⫾ 1.9 70.2 ⫾ 2 70 ⫾ 1.8

13.7 ⫾ 0.5 13 ⫾ 0.6 12.6 ⫾ 0.5

13.1 ⫾ 0.4 12.6 ⫾ 0.5 12.9 ⫾ 0.5

Peak E cm/s Baseline 6 months 12 months Peak A cm/s Baseline 6 months 12 months E/A ratio Baseline 6 months 12 months DT ms Baseline 6 months 12 months LVIRT ms Baseline 6 months 12 months

12.6 ⫾ 0.4 11.9 ⫾ 0.4 11.2 ⫾ 0.5

12.3 ⫾ 0.3 12.1 ⫾ 0.4 12.1 ⫾ 0.3

Abbreviations are: Peak E, peak of early diastolic flow velocity; peak A, peak of late diastolic flow velocity; E/A ratio, ratio of early to late diastolic flow; DT, deceleration time of E wave; LVIRT, left ventricular isovolumetric relaxation time.

176.4 ⫾ 7.6c 174.3 ⫾ 8.7 157.8 ⫾ 7.9d

160.5 ⫾ 7.3 160.3 ⫾ 6 164.1 ⫾ 7.6

0.2 ⫾ 4.4

1.68 ⫾ 3.9

⫺9.2 ⫾ 3.8a ⫺9.5 ⫾ 3.5b

2.8 ⫾ 2.3 3 ⫾ 3.2

11 (46)a

2 (7)

Abbreviations are: LVESD, left ventricular end-systolic diameter; LVEDD, left ventricular end-diastolic diameter; LAD, left auricular diameter; LVEF, left ventricular ejection fraction; IVST, interventricular septal thickness; PWT, posterior wall thickness; LVMI, left ventricular mass index; LVH, left ventricular hypertrophy. Data of LVMI changes at 12 months are presented in unadjusted form and when adjusted for age, baseline LVMI, time after grafting, and changes in systolic blood pressure, renal function and hemoglobin levels. a P ⬍ 0.01, b P ⬍ 0.05, c P ⬍ 0.1 vs. Placebo group d P ⬍ 0.05 vs. Baseline

were not significantly different (chi-square 4.76, P ⫽ 0.31) among the various groups studied. Genotype frequencies of both study groups, the healthy group and the unselected transplanted patients, were not significantly different from the predictions of the Hardy–Weinberg model (HW exact probability test, P ⫽ 0.30, P ⫽ 0.50, and P ⫽ 0.13, respectively). Several reports have demonstrated that DD patients have both higher angiotensin II levels and greater LVM than other genotypes [14–17]. Thus, we analyzed the effect of treatment in DD patients and in those with other genotype (ID/II) as a group, as has been performed in other studies [17, 31]. Table 4 shows clinical and echocardiographic data of the two treatment groups (placebo or lisinopril) in DD and ID/II patients separately. Pa-

Group A: Lisinopril N ⫽ 24

Group B: Placebo N ⫽ 28

73.1 ⫾ 4 78.5 ⫾ 4 74.7 ⫾ 4.3

69.3 ⫾ 4 75.8 ⫾ 4 71.4 ⫾ 3.6

65 ⫾ 3 72 ⫾ 4 69 ⫾ 3.8

71.4 ⫾ 4 73.8 ⫾ 3 76 ⫾ 4

1.17 ⫾ 0.9 1.15 ⫾ 0.9 1.14 ⫾ 0.9

1.02 ⫾ 0.8 1.06 ⫾ 0.7 1.01 ⫾ 0.8

236 ⫾ 11.5 215.3 ⫾ 10 236.6 ⫾ 13.8

241.8 ⫾ 13.6 232 ⫾ 11.7 235 ⫾ 17.2

103.1 ⫾ 9.4 101 ⫾ 9.7 96.4 ⫾ 6.6

99.3 ⫾ 7 98.9 ⫾ 6.5 98.6 ⫾ 6.4

tients with DD genotype receiving lisinopril showed a significant reduction of LVMI as compared with DD patients on placebo, after adjusting for age, baseline LVMI, time after grafting, and changes in systolic blood pressure, renal function and hemoglobin levels (⫺7.2 ⫾ 5.3 vs. 8.4 ⫾ 4.1%, P ⬍ 0.05). This effect could be due to a trend toward an increase of LVMI in DD patients on placebo at the end follow-up. The clinical consequence of the lisinopril effect on DD patients was a higher proportion of patients showing a reduction of LV mass ⱖ15% as compared with placebo (40 vs. 6.6%, P ⫽ 0.05). Likewise, DD patients receiving lisinopril also showed a trend toward a lower isovolumetric relaxation time at end of follow-up than those on placebo, after adjusting for the previously mentioned confounding variables (Table 4). On the other hand, ID/II patients under lisinopril also showed a trend toward a greater reduction of LVMI than those on placebo (Table 4). Interestingly, the beneficial effect of lisinopril in the DD group as compared with ID/II could not be due to a better control of blood pressure (mean blood pressure reduction DD, ⫺6.6 ⫾ 4.6 vs. ID/II, ⫺2 ⫾ 3.5, P ⫽ NS). DISCUSSION We demonstrated that a one-year treatment with lisinopril significantly decreased LVM in hypertensive renal transplant patients with LVH, and the highest change was for the posterior wall thickness. Moreover, the beneficial effect of lisinopril was independent of other factors

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Herna´ndez et al.: Lisinopril after renal transplantation Table 4. Clinical and echocardiographic data in patients with different genotypes at baseline, 6 and 12 months after treatment DD Patients (N ⫽ 25)

Age years Sex M/F Time after grafting months Systolic BP Baseline 6 months 12 months Diastolic BP Baseline 6 months 12 months MAP mm Hg Baseline 6 months 12 months MAP change % 12 months Serum creatinine mg/dL Baseline 6 months 12 months Hemoglobin levels change % 12 months LVMI g/m2 Baseline 6 months 12 months LVMI change % 12 months unadjusted adjusted LVIRT ms Baseline 6 months 12 months LVIRT change % 12 months unadjusted adjusted

ID and II Patients (N ⫽ 27)

Placebo N ⫽ 15

Lisinopril N ⫽ 10

Placebo N ⫽ 13

Lisinopril N ⫽ 14

49.6 ⫾ 3.9 10/5 73.5 ⫾ 8.2

48.5 ⫾ 4.5 6/4 67.5 ⫾ 11.6

51 ⫾ 3 8/5 81 ⫾ 7.8

46 ⫾ 3.2 10/4 56 ⫾ 3.8c

147.3 ⫾ 4.7 152.5 ⫾ 4.2 153.3 ⫾ 5.3

149.5 ⫾ 9.7 147 ⫾ 4 136.1 ⫾ 7d

148.5 ⫾ 2.8 154 ⫾ 4 147.8 ⫾ 4.6

146.3 ⫾ 3.2 144.5 ⫾ 6.5 143.8 ⫾ 4.6

91.5 ⫾ 3.4 89.68 ⫾ 1.8 89.4 ⫾ 3.4

85.8 ⫾ 6 84 ⫾ 4.5 78.2 ⫾ 3.4b

87.6 ⫾ 3.7 89.6 ⫾ 3.3 92.1 ⫾ 3.4

94 ⫾ 3.6 92.2 ⫾ 3.8 90.5 ⫾ 2.6

110 ⫾ 3 110.6 ⫾ 1.5 110.7 ⫾ 3.4 1.3 ⫾ 3.7

107 ⫾ 6.9 105 ⫾ 3.5 97.5 ⫾ 3.5a ⫺6.6 ⫾ 4.6

108 ⫾ 2.8 111.3 ⫾ 3 110.6 ⫾ 3.5 3.2 ⫾ 3.9

111.4 ⫾ 3.2 109.6 ⫾ 4.4 108.3 ⫾ 3 ⫺2 ⫾ 3.5c

1.5 ⫾ 0.1 1.27 ⫾ 0.9 1.36 ⫾ 0.1

1.48 ⫾ 0.1 1.45 ⫾ 0.1 1.34 ⫾ 0.8

1.5 ⫾ 0.1 1.43 ⫾ 0.1 1.47 ⫾ 0.1

1.47 ⫾ 0.9 1.5 ⫾ 0.1 1.5 ⫾ 0.9

0.7 ⫾ 0.4

⫺11.6 ⫾ 1.5a

⫺2 ⫾ 2

⫺11.5 ⫾ 2.3

157 ⫾ 8.6 154 ⫾ 6.6 168.2 ⫾ 9.4

168 ⫾ 13 177.8 ⫾ 12.4 154.7 ⫾ 12.6

164.5 ⫾ 12.5 168.3 ⫾ 10.5 159.4 ⫾ 12.6

182.4 ⫾ 9.4 172.3 ⫾ 12 160 ⫾ 8

7.8 ⫾ 3.2 8.4 ⫾ 4

⫺6.2 ⫾ 6b ⫺7.2 ⫾ 5.3b

⫺2.8 ⫾ 2.6 ⫺2.8 ⫾ 5.4

109.2 ⫾ 21 121 ⫾ 23 81.2 ⫾ 11.2d

96 ⫾ 8.4 83.4 ⫾ 6.4 90.1 ⫾ 8.5

102.2 ⫾ 11 112.3 ⫾ 9.7 106 ⫾ 7.7 11.5 ⫾ 10 16.5 ⫾ 11.4

⫺14.4 ⫾ 12d ⫺23 ⫾ 17.3d

⫺3.2 ⫾ 8 ⫺3.6 ⫾ 9

⫺11.4 ⫾ 5 ⫺11.4 ⫾ 5 99.2 ⫾ 8 89.6 ⫾ 6 105.8 ⫾ 7.5 8⫾6 8.4 ⫾ 8.2

Abbreviations are: BP, blood pressure; MAP, mean arterial pressure; LVMI, left ventricular mass index; LVIRT, left ventricular isovolumetric relaxation time. Data of LVMI and LVIRT changes are shown in unadjusted form and when adjusted for age, baseline LVMI and LVIRT, time after grafting, and changes in systolic blood pressure, renal function and hemoglobin levels. DD patients: Lisinopril vs. Placebo: aP ⫽ 0.02; bP ⬍ 0.05; dP ⬍ 0.1 ID/II patients: Lisinopril vs. Placebo: cP ⬍ 0.01

known to affect LVM such as age, blood pressure, baseline LVM, time after grafting, renal function, and hemoglobin levels. In addition, the beneficial effect of lisinopril was more evident in DD patients. In order to minimize coexistent abnormalities, we only included nondiabetic patients with an acceptable renal function. We used 10 mg/day of lisinopril, which is the minimal reported dose that reduces LVM in hypertensive patients [32]. No drugrelated side effects were observed in the cohort completing the study, although five patients were excluded in the initial evaluation as a consequence of renal function impairment. To our knowledge, this is the first randomized placebo-controlled study to investigate the efficacy of lisinopril on LVM in renal transplant patients with hypertension and LVH. This condition may be a major cardiovascular risk factor in these patients, as has been

demonstrated in the general population [3]. Thus, a reduction of LVM indeed emerges as a desirable goal in renal transplant patients. Since ACEIs may induce acute renal failure even without renal artery stenosis [24, 33], this controlled study was performed in a single-blind design. The increase in serum creatinine has been related to changes in the glomerular hemodynamics, with a transient decrease in glomerular filtration rate. As expected, a greater reduction in hemoglobin levels was observed in patients receiving lisinopril, but it had no clinical significance. This side effect is very common with the use of ACEIs [34] and may have a deleterious effect on LVMI [35]. However, a higher reduction of LVM was observed in this group, suggesting that blockade of local angiotensin II by lisinopril was more important than the effect on hemoglobin levels.

Herna´ndez et al.: Lisinopril after renal transplantation

Both groups received a similar proportion of antihypertensive drugs other than lisinopril during the follow-up. Although blood pressure was well controlled during the study, patients receiving lisinopril showed a trend toward a greater reduction of blood pressure at the end of the study. Thus, this effect may have contributed to the beneficial effect of lisinopril on LVMI. However, after adjusting for blood pressure changes, lisinopril induced a significant reduction of LVMI, which was not observed in the placebo group. In addition, no correlation between LVM and blood pressure changes during the study was observed (data not shown). This finding is in agreement with previous observations both in hypertensive and normotensive humans, showing that the ACEIs are able to induce regression of LVM by mechanisms that are independent of blood pressure changes [36, 37]. Previous studies have clearly demonstrated that angiotensin II participates in the development of myocardial hypertrophy, regardless of changes in arterial pressure and cardiac workload [11, 38]. ACEIs may antagonize the growth-promoting influence of angiotensin II, and the local blockade of this system may be more important than the circulating one. Indeed, ACEIs, given in very low doses that do not affect blood pressure, prevented the development of cardiac hypertrophy in pressureoverloaded rats [39]. Another possible mechanism is that blockade of local angiotensin II by lisinopril increases aortic compliance, leading to LVH reversal, as has been recently demonstrated in hypertensive patients [36]. Finally, blockade of the renin-angiotensin system could also improve sympathovagal imbalance, modulating the reactivity and release of cathecolamines, which could have trophic influences on myocardial growth [40]. In this study, overall diastolic function was similar in both groups during follow-up, and the E/A ratio did not change. London et al were also unable to observe changes in diastolic function after treatment with ACEIs for one year in uremic patients [37]. It is possible that a longer period of treatment and/or a greater reduction in LVM are needed to induce significant changes in diastolic function. This is supported by recent data demonstrating that the improvement of diastolic function with ACEIs was related to reduction in ventricular mass and not to a decrease in mean arterial pressure or improvement in aortic compliance [36, 41]. An I/D polymorphism of the ACE gene has been shown to be a genetic factor influencing both circulating and tissue ACE levels. ACE activity is higher in DD patients than other genotypes, and these subjects have a greater cardiovascular risk [13]. In fact, in our DD patients receiving placebo for one year, LVMI showed a mean increase of 8.4%, which contrasts with a 2.8% decrease in ID/II patients on placebo (these differences did not reach statistical significance possibly as a consequence of the sample size). A similar effect had been

895

observed in our previous studies [18]. Thus, the beneficial effect of the ACEIs may be under genetic influence. We analyzed individual evolution of LVM in patients with different genotypes after treatment with lisinopril. DD patients receiving lisinopril showed a significant reduction of LVM as compared with patients on placebo, after adjusting for other confounder covariates. Patients with ID or II genotype also showed a higher decrease of LVMI as compared with those with placebo, although a greater sample size may be needed to reach statistical significance. Finally, DD patients on lisinopril showed a trend to lower isovolumetric relaxation time at the end of follow-up as compared with DD patients on placebo. Similar to our findings, Sasaki et al observed that changes in LVMI and diastolic function were significantly greater in DD hypertensive patients receiving enalapril [21]. Other authors have also shown a greater antiproteinuric effect of ACEIs in renal patients with the DD genotype, suggesting a more intensive vasodilatation effect in this group [19, 42]. As a higher availability of local angiotensin II in DD patients would be expected, the antiproliferative and vasodilator effect of ACEIs might be more prominent in renal transplant patients with DD genotype, thus inducing a greater reduction of LVM. In contrast to our study, a major beneficial effect of ACEIs has not been found in uremic or diabetic patients with DD genotype [31, 43, 44]. It is possible that other factors like volume overload, anemia, endocrine disorders, or high parathyroid hormone levels present in these patients may obscure differences between genotypes. In addition, ethnic heterogeneity may play an important role in the response to ACEIs in patients with different genotypes of the ACE gene. A recent meta-analysis, showing different evolution of diabetic nephropathy between Caucasian and Asian DD patients, supports this hypothesis [45]. Cyclosporine clearly stimulates both circulating and local renin-angiotensin systems, as evidenced by its activation of different components of this system in multiple tissues [46]. An activated renin-angiotensin system may help to separate better the consequences of the ACE polymorphism in renal transplant patients. In other words, the ACE DD genotype may have a permissive role in the development of LVH when the cardiac growth machinery is stimulated. Therefore, a more pronounced effect of ACEIs on LVM regression might be expected in DD transplant patients. In conclusion, lisinopril decreases LVM in renal transplant patients with hypertension and LVH, regardless of other clinical data that interact on ventricular mass. In addition, the I/D ACE genotype may predict the extent to which LVM will be lowered by lisinopril. This effect may be important in targeting prophylactic interventions in these patients.

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Herna´ndez et al.: Lisinopril after renal transplantation

ACKNOWLEDGMENTS This study was supported by grant (PI 71/98) from Consejeria de Sanidad y Consumo (Gobierno de Canarias), Fundacio´n Canaria de Investigacio´n y Salud (FUNCIS), and grant 96/0857 of the Spanish Ministry of Health (FIS). The authors are grateful to Dr. Armas and the house staff for their cooperation and help. The authors also acknowledge Vita S.A. (Barcelona, Spain) for providing the tablets for this study, but there is no other link or conflict of interest between the authors and any pharmaceutical company. Reprint requests to Domingo Herna´ndez, M.D., Urbanizacio´n San Diego, 51, E-38208 La Laguna, Tenerife, Canary Islands, Spain. E-mail: [email protected]

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19.

REFERENCES 1. First MR: Long-term complications after transplantation. Am J Kidney Dis 22:477–486, 1993 2. US Renal Data System: USRDS 1994: Annual Data Report, The National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases: Causes of death. Am J Kidney Dis 24(Suppl 2):S88–S95, 1994 3. Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP: Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N Engl J Med 322:1561–1566, 1990 4. Lipkin GW, Tucker B, Giles M, Raine AE: Ambulatory blood pressure and left ventricular mass in cyclosporin- and non-cyclosporine-treated renal transplant recipients. J Hypertens 11:439– 442, 1993 5. Fishel RS, Eisenberg S, Shai S-Y, Redden RA, Bernstein KE, Berk BC: Glucocorticoids induce angiotensin-converting enzyme expression in vascular smooth muscle. Hypertension 25:343–349, 1995 6. Ventura HO, Lavie CJ, Messerli FH, Valentino V, Smart FW, Stapleton DD, Ochsner JL: Cardiovascular adaptation to cyclosporine-induced hypertension. J Hum Hypertens 8:233–237, 1994 7. Sadoshima J-I, Izumo S: Molecular characterization of angiotensin II-induced hypertrophy of cardiac myocites and hyperplasia of cardiac fibroblasts: Critical role of the AT1 receptor subtype. Circ Res 73:413–423, 1993 8. Brilla CG, Zhou G, Matsubara L, Weber KT: Collagen metabolism in cultured adult rat cardiac fibroblasts: Response to angiotensin II and aldosterone. J Mol Cell Cardiol 26:809–820, 1994 9. Lindpaintner K, Ganten D: The cardiac renin-angiotensin system: An appraisal of present experimental and clinical evidence. Circ Res 68:905–921, 1991 10. Dahlof B, Pennert K, Hansson L: Reversal of left ventricular hypertrophy in hypertensive patients: A meta-analysis of 109 treatment studies. Am J Hypertens 5:95–110, 1992 11. Schmieder RE, Schlaich MP, Klingbeil AU, Martus P: Update on reversal of left ventricular hypertrophy in essential hypertension (a meta-analysis of all randomized double-blind studies until December 1996). Nephrol Dial Transplant 13:564–569, 1998 12. Samani NJ, Thompson JR, O’Toole L, Channer K, Woods KL: A meta-analysis of the association of the deletion allele of the angiotensin-converting enzyme gene with myocardial infarction. Circulation 94:708–712, 1996 13. Butler R, Morris AD, Struthers AD: Angiotensin-converting enzyme gene polymorphism and cardiovascular disease. Clin Sci 93:391–400, 1997 14. Schunkert H, Hense H-W, Holmer SR, Stender M, Perz S, Keil U, Lorell BH, Iegger GAJ: Association between a deletion polymorphism of the angiotensin-converting-enzyme gene and left ventricular hypertrophy. N Engl J Med 330:1634–1638, 1994 15. Iwai N, Ohimichi N, Nakamura Y, Kinoshita M: DD-genotype of the angiotensin-converting enzyme gene is a risk factor for left ventricular hypertrophy. Circulation 90:2622–2628, 1994 16. Perticone F, Ceravolo R, Cosco Traspasso M, Zingone A, Malatesta P, Perroti N, Tramontano D, Mattioli PL: Deletion polymorphism of angiotensin-converting enzyme gene and left ven-

20.

21.

22.

23.

24. 25.

26. 27. 28. 29.

30. 31.

32.

33.

34.

tricular hypertrophy in southern Italian patients. J Am Coll Cardiol 29:365–369, 1997 Osono E, Kurihara S, Hayama N, Sakurai Y, Ohwada K, Onoda N, Takeuchi M, Tomizawa T, Komaba Y, Hashimoto K, Matsunobu S, Yoneshima H, Iino Y: Insertion/deletion polymorphism in intron 16 of the ACE gene and left ventricular hypertrophy in patients with end-stage renal disease. Am J Kidney Dis 32:725–730, 1998 Hernandez D, Lacalzada J, Rufino M, Torres A, Marti´n N, Barraga´n A, Barrios Y, Maci´a M, De Bonis E, Lorenzo V, Rodri´guez A, Gonza´lez-Posada JM, Salido E: Prediction of left ventricular mass changes after renal transplantation by polymorphism of the angiotensin-converting-enzyme gene. Kidney Int 51:1205–1211, 1997 Moriyama T, Kitamura H, Ochi S, Izumi M, Yokoyama K, Yamauchi A, Ueda N, Kamada T, Imai E: Association of angiotensin Iconverting enzyme gene polymorphism with susceptibility to antiproteinuric effect of angiotensin I-converting enzyme inhibitors in patients with proteinuria. J Am Soc Nephrol 6:1674–1678, 1995 Van Essen GG, Rensma PL, De Zeeuw D, Sluiter WJ, Scheffer H, Apperloo D, De Jong PE: Association between angiotensinconverting-enzyme gene polymorphism and failure of renoprotective therapy. Lancet 347:94–95, 1996 Sasaki M, Oki T, Iuchi A, Tabata H, Yamada H, Manabe K, Fukuda K, Abe M, Ito S: Relationship between the angiotensin converting enzyme gene polymorphism and the effects of enalapril on left ventricular hypertrophy and impaired diastolic filling in essential hypertension: M-mode and pulsed Doppler echocardiographic studies. J Hypertens 14:1403–1408, 1996 Gonza´lez-Posada JM, Castro MCG, Losada M, Lorenzo V, Torres A, Herna´ndez D, Maceira B, Salido E: Monoclonal analysis of fine-needle aspiration biopsy in kidney allografts. Nephrol Dial Transplant 5:226–231, 1990 Lancaster SG, Todd PA: Lisinopril: A preliminary review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in hypertension and congestive heart failure. Drugs 35:646– 649, 1988 Mimran A, Ribstein J, Ducailar G: Converting enzyme inhibitors and renal function in essential and renovascular hypertension. Am J Hypertens 4(Suppl 2):S7–S14, 1991 Koren MJ, Devereux RB, Casale PN, Savage DD, Laragh JH: Relation of left ventricular mass and geometry to morbidity and mortality in uncomplicated essential hypertension. Ann Intern Med 114:345–352, 1991 Devereux R, Reichek N: Echocardiographic determination of left ventricular mass in man: Anatomic validation of the method. Circulation 55:613–618, 1977 Krumholz HM, Larson M, Levy D: Prognosis of left ventricular geometric patterns in the Framingham heart study. J Am Coll Cardiol 25:879–884, 1995 Teichholz L, Kreulen T, Herman M, Gonlin R: Problems in echocardiographic angiographic correlations in the presence or absence of asynergy. Am J Cardiol 37:7–11, 1976 Lindpaintner K, Pfeffer MA, Kreutz R, Stamper MJ, Grodstein F, Lamotte F, Buring J, Hennekens CH: A prospective evaluation of an angiotensin-converting-enzyme gene polymorphism and the risk of ischemic heart disease. N Engl J Med 332:706–711, 1995 Davis LG, Dibner MD, Battey JF: Basic Methods in Molecular Biology. New York, Elsevier, 1986, pp 44–46 Canella G, Paoletti E, Barocci S, Massarino F, Delfino R, Ravera G, Maio GD, Nocera A, Patrone P, Rolla D: Angiotensin-converting enzyme gene polymorphism and reversibility of uremic left ventricular hypertrophy following long-term antihypertensive patients. Kidney Int 54:618–626, 1998 Esper RJ, Burrieza OH, Cacharro´n JL, Fa´bregues G, Baglivo HP: Left ventricular mass regression and diastolic function improvement in mild and moderate hypertensive patients treated with lisinopril. Cardiology 83:76–81, 1993 Traindl O, Falger S, Reading S, Banyai Liebisch B, Gisinger J, Templ E, Mayer G, Koverik J: The effects of lisinopril on renal function in proteinuric renal transplant recipients. Transplantation 55:1309–1313, 1993 Vlahakos DV, Canzanello VJ, Madaio MP, Madias NE: Enala-

Herna´ndez et al.: Lisinopril after renal transplantation

35.

36.

37.

38.

39.

40.

pril-associated anemia in renal transplant recipients treated for hypertension. Am J Kidney Dis 17:199–205, 1991 Amann K, Rychli´k I, Miltenberger-Milteny G, Ritz E: Left ventricular hypertrophy in renal failure. Kidney Int 54(Suppl 68):S78–S85, 1998 Shimamoto H, Shimamoto Y: Lisinopril reverses left ventricular hypertrophy through improved aortic compliance. Hypertension 28:457–463, 1996 London GM, Pannier B, Guerin AP, Marchais SJ, Safar ME, Cuche J-L: Cardiac hypertrophy, aortic compliance, peripheral resistance, and wave reflection in end-stage renal disease: Comparative effects of ACE inhibition and calcium channel blockade. Circulation 90:2786–2796, 1994 Schmieder RE, Langenfeld MRW, Friedrich A, Schobel HP, Gatzka CD, Weihprecht H: Angiotensin II related to sodium excretion modulates left ventricular structure in human essential hypertension. Circulation 94:1304–1309, 1996 Linz W, Shaper J, Wiemer G, Albus U, Scho¨lken S: Ramipril prevents left ventricular hypertrophy with myocardial fibrosis without blood pressure reduction: A one year study in rats. Br J Pharmacol 107:970–975, 1992 Petretta M, Bonaduce D, Marciano F, Bianchi V, Valva G, De Apicella C, Luca N, Gisoni P: Effect of 1 year of lisinopril

41. 42.

43.

44.

45. 46.

897

treatment on cardiac autonomic control in hypertensive patients with left ventricular hypertrophy. Hypertension 27:330–338, 1996 Oren S, Grossman E, Frohlich ED: Reduction in left ventricular mass in patients with systemic hypertension treated with enalapril, lisinopril, or fosinopril. Am J Cardiol 77:93–96, 1996 Yoshida H, Mitarai T, Kawamura T, Kitajima T, Miyazaki Y, Nagasawa R, Kawaguchi Y, Kubo H, Ichikawa I, Sakai O: Role of the deletion of polymorphism of the angiotensin converting enzyme gene in the progression and therapeutic responsiveness of IgA nephropathy. J Clin Invest 96:2162–2169, 1995 Penno G, Chaturvedi N, Talmud PJ, Cotroneo P, Manto A, Nannipieri M, Luong LA, Fuller JH: Effect of angiotensin-converting enzyme gene polymorphism on progression of renal disease and the influence of ACE inhibition in IDDM patients: Findings from the EUCLID randomized controlled trial: EURODIAB controlled trial of lisinopril in IDDM. Diabetes 47:1507–1511, 1998 Jacobsen P, Rossing K, Rossing P, Tarnow L, Mallet C, Poirier O, Cambien F, Parving HH: Angiotensin converting enzyme gene polymorphism and ACE inhibition in diabetic nephropathy. Kidney Int 53:1002–1006, 1998 Tarnow L, Gluud C, Parving H-H: Diabetic nephropathy and the insertion/deletion polymorphism of the angiotensin-converting enzyme gene. Nephrol Dial Transplant 13:1125–1130, 1998 Lee DBN: Cyclosporine and the renin-angiotensin axis. Kidney Int 52:248–260, 1997