Measurable Urinary Albumin Predicts Cardiovascular Risk Among Normoalbuminuric Patients with Type 2 Diabetes

CLINICAL EPIDEMIOLOGY www.jasn.org

Measurable Urinary Albumin Predicts Cardiovascular Risk among Normoalbuminuric Patients with Type 2 Diabetes

† Piero Ruggenenti,* Esteban Porrini,* Nicola Motterlini,* Annalisa Perna,* ‡ ‡ Aneliya Parvanova Ilieva,* Ilian Petrov Iliev,* Alessandro Roberto Dodesini, Roberto Trevisan, | Antonio Bossi,§ Giuseppe Sampietro, Enrica Capitoni,¶ Flavio Gaspari,* Nadia Rubis,* † Bogdan Ene-Iordache,* and Giuseppe Remuzzi,* for the BENEDICT Study Investigators

*Clinical Research Center for Rare Diseases, “Aldo & Cele Daccò,” Mario Negri Institute for Pharmacological Research, Bergamo, Italy; †Units of Nephrology and ‡Diabetology and ¶Research Foundation, Azienda Ospedaliera, “Ospedali Riuniti di Bergamo,” Bergamo, Italy; §Unit of Diabetology, Treviglio Hospital, Treviglio, Italy; and |Epidemiological Observatory, “Azienda Sanitaria Locale della Provincia di Bergamo,” Bergamo, Italy

ABSTRACT Micro- or macroalbuminuria is associated with increased cardiovascular risk factors among patients with type 2 diabetes, but whether albuminuria within the normal range predicts long-term cardiovascular risk is unknown. We evaluated the relationships between albuminuria and cardiovascular events in 1208 hypertensive, normoalbuminuric patients with type 2 diabetes from the BErgamo NEphrologic Diabetes Complication Trial (BENEDICT), all of whom received angiotensin-converting enzyme inhibitor (ACEI) therapy at the end of the trial and were followed for a median of 9.2 years. The main outcome was time to the first of fatal or nonfatal myocardial infarction; stroke; coronary, carotid, or peripheral artery revascularization; or hospitalization for heart failure. Overall, 189 (15.6%) of the patients experienced a main outcome event (2.14 events/100 patient-years); 24 events were fatal. Albuminuria independently predicted events (hazard ratio [HR], 1.05; 95% confidence interval [CI], 1.02–1.08). Second-degree polynomial multivariable analysis showed a continuous nonlinear relationship between albuminuria and events without thresholds. Considering the entire study population, even albuminuria at 1–2 mg/min was significantly associated with increased risk compared with albuminuria ,1 mg/min (HR, 1.04; 95% CI, 1.02–1.07). This relationship was similar in the subgroup originally randomly assigned to non-ACEI therapy. Among CLINICAL EPIDEMIOLOGY those originally receiving ACEI therapy, however, the event rate was uniformly low and was not significantly associated with albuminuria. Taken together, among normoalbuminuric patients with type 2 diabetes, any degree of measurable albuminuria bears significant cardiovascular risk. The association with risk is continuous but is lost with early ACEI therapy.

J Am Soc Nephrol 23: 1717–1724, 2012. doi: 10.1681/ASN.2012030252

Patients with type 2 diabetes mellitus and micro- or macroalbuminuria (i.e., a urinary albumin excre- Received March 9, 2012. Accepted July 11, 2012. – m . m tion [UAE] rate of 20 200 g/min or 200 g/ P.R. and E.P. contributed equally to this work. min, respectively) have a risk for cardiovascular – Published online ahead of print. Publication date available at death that is 2 12 times that observed in patients www.jasn.org. with less albuminuria.1,2 This largely accounts for the excess cardiovascular mortality observed in pa- Correspondence: Dr. Giuseppe Remuzzi, Mario Negri Institute for Pharmacological Research, Centro Anna Maria Astori Science tients with diabetes compared with age-matched and Technology Park, Kilometro Rosso Via Stezzano, 87. 24126 persons without the disease.1,3,4 Conversely, dia- Bergamo, Italy. Email: [email protected], manuela. betic patients with UAE ,20 mg/min (the upper [email protected] limit of what is generally considered the normal Copyright © 2012 by the American Society of Nephrology

J Am Soc Nephrol 23: 1717–1724, 2012 ISSN : 1046-6673/2310-1717 1717 CLINICAL EPIDEMIOLOGY www.jasn.org range) are supposed to have a cardiovascular risk close to that causes. Overall, 212 major cardiovascular events were ob- of the general population.5–7 However, this cutoff was intro- served in 189 patients. duced aprioriin clinical use and research based on the observation that 95% of “normal” individuals had excretion rates Primary Composite End-Point below that limit.8 Whether this value reflects a real threshold Overall, first onset of a component of the composite end point for cardiovascular risk or whether any measurable amount of of major cardiovascular events was observed in 189 of the albuminuriabearsasignificant risk, even within the normal 1208 randomly assigned participants (15.6%). In 24 cases range, is unknown, particularly in the diabetic population. (12.7%), the event was fatal. In addition to 3 sudden-death Whether there is a level for albuminuria that differentiates events (1.6%), there were 105 (55.6%), 39 (20.6%), and 31 patients who need cardioprotective intervention from those (16.4%) events related to coronary, cerebrovascular, or pe- with a low risk who are unlikely to be affected by any treat- ripheral artery disease, respectively, and 11 hospitalizations ment is also elusive. This is a major health issue because per- (5.8%) due to worsening of congestive heart failure (Table 2). sons with normoalbuminuria account for the large majority of Throughout the whole observation period, there were 2.14 the diabetic population, and promptly identifying those at risk patients with the end point event every 100 patients per year. and providing them with preventive strategies before they Overall, there were 55 end point events (4 fatal) during progress to more severe and possibly irreversible stages of BENEDICT-A and 133 (20 fatal) during the extension study. the disease might have major clinical implications. Compared with patients without cardiovascular events, To address the above issues, we evaluated a large cohort of those progressing to the end point were older and more patients with type 2 diabetes and baseline UAE ,20 mg/min frequently male, more frequently reported a cardiovascular who were included in the BErgamo NEphrologic Diabetes event before study inclusion, and had more albuminuria and 9 Complication Trial (BENEDICT-A) and eventually followed higher HbA1c and serum LDL-to-HDL cholesterol ratio for approximately 10 years. In BENEDICT, at similar BP con- (Table 1). Other baseline characteristics (including BP; control, angiotensin-converting enzyme inhibitor (ACEI) treat- comitant antihypertensive, antidiabetic, and lipid-lowering ment with trandolapril alone or in combination with the treatment; and whether patient was allocated to ACEI therapy) nondihydropyridine calcium-channel blocker verapamil were similar between groups with or without end point events. halved the incidence of microalbuminuria compared with verapamil alone or placebo.9,10 Baseline Characteristics and Cardiovascular Risk We primarily sought to address whether and to what At univariable Cox proportional hazards regression analysis, extent baseline albuminuria was associated with long-term older age; male sex; a longer duration of diabetes; previous incidence of fatal and nonfatal cardiovascular events in the cardiovascular events; and higher LDL-to-HDL cholesterol above population. Secondarily, we explored whether earlier ratio, creatinine levels, and albuminuria levels were signifi- treatment with an ACEI translated into more effective cantly associated with a higher risk for new-onset cardiovas- cardioprotection in the long term and whether there was a cular events (Table 3). At multivariable analysis, albuminuria threshold for baseline albuminuria above which this effect was significantly associated with risk for cardiovascular events became apparent. (hazard ratio [HR], 1.06; 95% confidence interval [CI], 1.02– 1.08). On average, for 1 mg/min excess of albuminuria at baseline there was a 6% risk for progression to the end point on RESULTS follow-up. Other independent predictors of events were older age, male sex, previous cardiovascular events, higher HbA1c Baseline Characteristics levels, and higher LDL-to-HDL cholesterol ratio. No signifi- Median UAE at inclusion was 5.24 (range, 0.44–19.85) mg/min. cant interaction between albuminuria and the other baseline UAE exceeded 14 mg/min in only 10% of participants (Table 1). covariates was observed. The assumption of proportionality Baseline characteristics of study participants were similar to was not violated (Schoenfeld residuals: P=0.26). those of the 139 patients who did not enter the BENEDICT The second-degree polynomial transformation of alb- extension study, with the exception of a higher prevalence of uminuria described the relationship with the cardiovascular men, lower hemoglobin A1c (HbA1c), and lower LDL-to-HDL end point with lower deviance (best goodness of fit) than the cholesterol ratio in included participants (Supplemental Table first degree (logarithmic) transformation (931.32 versus 1). A similar proportion of patients originally randomly as- 934.27, respectively). Modeling baseline albuminuria by sec- signed in BENEDICT to trandolapril or non-ACEI therapy ond-degree polynomial transformation in the multivariable was receiving ACEI (87.5%) or statin (50.5%) therapy during analysis showed a continuous nonlinear relationship between the extension study. albuminuria and cardiovascular events (Figure 1). A significant excess risk for progression to the end point was already Outcomes evident for a UAE ranging from 1 to 2 mg/min compared with During a median follow-up of 9.12 (interquartile range, 6.16– an excretion ,1 mg/min (HR, 1.04; 95% CI, 1.02–1.07) taken 9.86) years, 168 patients died, 37 (22%) of cardiovascular as the reference value. The risk for events compared with the

1718 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 1717–1724, 2012 www.jasn.org CLINICAL EPIDEMIOLOGY

Table 1. Baseline characteristics of the overall study group and of patients with or without major cardiovascular events during follow-up Characteristic Overall Without Events With Events P Valuea Patients, n (%) 1208 1019 (84.4) 189 (15.6) Age (yr) 62.3368.05 61.8568.12 64.9267.15 ,0.001 Men, n (%) 638 (52.8) 521 (51.1) 117 (61.9) 0.004 History of cardiovascular events 52 (4.3) 34 (3.3) 18 (9.5) ,0.001 Known duration of diabetes (yr) 6 (3–11) 6 (2–11) 7 (3–12) 0.18 Smoking status, n (%) Never smoked 700 (57.9) 600 (58.9) 100 (52.9) 0.20 Former smoker 362 (30.0) 302 (29.6) 60 (31.7) Current smoker 146 (12.1) 117 (11.5) 29 (15.3) Body mass index (kg/m2) 29.0864.72 29.1664.72 28.6664.68 0.19 Trough BP (mmHg) Systolic 150.82614.16 150.77614.17 151.10614.16 0.77 Diastolic 87.4667.63 87.6067.47 86.6068.44 0.13 Mean 108.5868.35 108.6668.21 108.1769.07 0.45 Glycosylated hemoglobin (%) 5.7961.37 5.7361.36 6.1661.37 ,0.001 Glucose (mg/dl) 161.34647.05 160.00646.67 168.52648.57 0.03 Cholesterol (mg/dl) Total 209.78636.74 209.05637.21 213.67633.93 0.11 HDL 46.94612.06 47.29612.11 45.06611.65 0.02 LDL 162.75636.05 161.60636.36 168.82633.81 0.01 Triglycerides 125 (91–181) 124 (91–177) 134 (91–194) 0.12 Serum creatinine (mg/dl) 0.9060.16 0.9060.15 0.9360.17 0.02 Uric acid (mg/dl) 4.0961.18 5.0161.19 5.0061.13 0.90 Albuminuria (mg/min) 5.24 (3.5–9.3) 5.09 (3.5–8.7) 7.08 (3.7–11.6) ,0.001 Allocation to study treatments, n (%) Placebo 302 (25.0) 258 (25.3) 44 (23.3) 0.15 Verapamil 303 (25.1) 243 (23.8) 60 (31.7) Trandolapril 302 (25) 259 (25.4) 43 (22.8) Verapamil plus trandolapril 301 (24.9) 259 (25.4) 42 (22.2) Use of antihypertensive agents, n (%)b 664 (55.0) 556 (54.6) 108(57.1) 0.32 Number of antihypertensive agentsc 2(1–2) 2 (1–2) 2 (1–3) 0.28 Antilipidemic agents, n (%) 138 (11.4) 119 (11.7) 19 (10.0) 0.30 Aspirin, n (%) 28 (2.3) 17 (1.7) 11 (5.8) 0.002 Duration of follow-up (yr) 9.12 (6.16–9.86) 9.09 (6.0–9.81) 9.28 (7.7–10.10) 0.03 Data are numbers, percent values, mean 6 SD, or median (interquartile range). aWith versus without events. bDifferent from the study treatments. cIncluding the study treatments. reference albuminuria level tended to plateau (HR, 2.49; 95% were analyzed (data not shown). Unlike BP,albuminuria at the CI, 1.56–4.00) between albuminuria values of 13 and 14 mg/ end of BENEDICT—that is, at baseline of the extension min (Figure 1 and Supplemental Table 2). Results were similar study—predicted cardiovascular events on subsequent fol- when baseline albuminuria was modeled with a spline func- low-up at univariate or multivariable analyses (odd ratios, 1.41 tion (data not shown). Cardiovascular risk at higher levels of [95% CI, 1.15–1.74], P=0.001, and 1.29 [95% CI, 1.04–1.60], albuminuria was not considered because of the too small P=0.001, respectively). Consistently, patients with persistent low- number of patients and events. range compared with those with persistent high-range normoalbuminuria (i.e., albuminuria ,1SDor.1SDofthemeanat Sensitivity Analyses baseline or at BENEDICT last visit, respectively) had a three-fold Relationships between albuminuria and outcomes similar to lower incidence of cardiovascular events during the extension those described in the whole study group were observed when study (8% versus 24%; P=0.027). the analyses were restricted to low-risk patients without a history of cardiovascular events at inclusion or with persistent Cardiovascular Risk According to ACEI Therapy normoalbuminuria throughout BENEDICT, as well as when Baseline characteristics of patients who had been originally cardiovascular events were considered independent of revas- allocated to ACEI therapy with trandolapril alone or combined cularizations and amputations or when only coronary events with verapamil in BENEDICTwere similar to those of patients

J Am Soc Nephrol 23: 1717–1724, 2012 Albuminuria and Cardiovascular Disease 1719 CLINICAL EPIDEMIOLOGY www.jasn.org

Table 2. Patients with first-onset fatal or nonfatal major to the end point compared with 77 receiving non-ACEI cardiovascular events (primary end point) therapy, and the incidence of events was significantly lower Event Patients (n) in those with ACEI (9.5% versus 12.7%; P=0.043). Coronary heart disease (n=105) As in the study group considered as a whole, a continuous Unstable angina pectoris 14 significant incremental risk for cardiovascular events for each Unstable angina pectoris + stenting/revascularization 40 1 mg/min increase in albuminuria at baseline was observed in Acute myocardial infarction 46 patients originally randomly assigned to non-ACEI therapy Acute myocardial infarction + stenting/revascularization 5 considered separately. Again, in this group a significant in- Congestive heart failure 11 crease was already evident for a UAE ranging from 1 to 2 Stroke or transient ischemic attack (n=39) mg/min compared with ,1 mg/min (HR, 1.02; 95% CI, Stroke 24 1.01–1.074) taken as the reference value. The risk for events Transient ischemic attack 15 tended to plateau (HR, 3.15; 95% CI, 1.76–5.63) between Sudden death 3 albuminuria values of 13 and 14 mg/min (Figure 2 and Sup- Peripheral vascular disease (n=31) Severe carotid obstruction 5 plemental Table 2). Of note, however, in patients originally Severe carotid obstruction + stenting or angioplasty 8 randomly assigned to ACEI therapy, the risk for major cardio- Severe peripheral obstruction 8 vascular events was uniformly low at any considered level of Severe peripheral obstruction + stenting or bypass 10 albuminuria at baseline, and no significant association was surgery observed between any increment in albuminuria and cardio- Total 189 vascular events during the whole study period (Figure 2 and Supplemental Table 2).

Table 3. Univariate and multivariable Cox proportional hazards regression DISCUSSION analysis of covariates versus the primary end point (major cardiovascular events) Univariate Analysis Multivariable Analysis In this longitudinal observational study of Covariates . HR (95% CI) P Value OR (95% CI) P Value 1000 patients with type 2 diabetes and normoalbuminuria, we found a con- Age 1.05 (1.03–1.07) ,0.001 1.06 (1.04–1.08) ,0.001 tinuous relationship between UAE rate at Female versus male 1.46 (1.09–1.96) 0.01 1.58 (1.07–2.32) 0.02 Duration of diabetes 1.02 (1.002–1.043) 0.03 0.99 (0.97–1.02) 0.88 inclusion and incidence of major cardio- History of cardiovascular 3.02 (1.85–4.91) ,0.001 2.97 (1.79–4.93) ,0.001 vascular events during approximately 10 disease years of follow-up. For each 1 mg/min ex- Smoker status 1.38 (0.93–2.04) 0.11 1.25 (0.81–1.92) 0.31 cess in baseline albuminuria, there was a Body mass index 0.96 (0.95–1.009) 0.16 0.99 (0.96–1.03) 0.65 progressive incremental risk for cardiovas- Mean BP 0.99 (0.97–1.008) 0.31 0.98 (0.96–1.00) 0.05 cular events up to a UAE of 13–14 mg/min. a – – HbA1c 2.87 (1.56 5.27) 0.001 3.17 (1.55 6.48) 0.002 Above this level, the number of patients LDL-to-HDL cholesterol 1.93 (1.26– 2.94) 0.002 2.03 (1.21–3.39) 0.007 was too small to evaluate any relationship a ratio between albuminuria and events. The in- – – Triglycerides 1.001 (0.999 1.003) 0.07 1.00 (0.99 1.002) 0.90 cremental risk for events, compared with Serum creatininea 2.54 (1.10–5.85) 0.03 0.86 (0.31–2.42) 0.78 a – , – the reference risk observed with a UAE UAE 1.055 (1.026 1.086) 0.001 1.05 (1.02 1.08) 0.002 , m Uric acid 1.002 (0.891–1.127) 0.97 1.01 (0.88–1.16) 0.90 1 g/min, was already evident at an ex- m Allocation to ACEI 0.818 (0.614–1.089) 0.17 0.84 (0.63–1.13) 0.25 cretion rate ranging from 1 to 2 g/min. Use of lipid-lowering therapy 0.857 (0.533–1.377) 0.52 0.79 (0.48–1.29) 0.34 The incremental incidence of cardiovascu- OR, odds ratio. lar events observed for each 1 mg/min ex- aVariables log-transformed. cess in baseline UAE was significant in the study group as a whole as well as in the subgroup of patients who had been origi- randomly assigned to non-ACEI therapy with verapamil alone nally randomly assigned in BENEDICT to non-ACEI therapy or placebo.11 Eighty-four of the 603 patients (13.9%) originally with verapamil alone or placebo. Of note, however, the asso- randomly assigned to ACEI therapy had at least one major ciation between albuminuria and events was not significant in cardiovascular event compared with 105 of the 605 patients patients who had been randomly assigned to ACEI therapy (17.4%) randomly assigned to non-ACEI therapy (P=0.08). with trandolapril alone or in combination with verapamil. During BENEDICT, 27 (4.5%) participants receiving ACEI Actually, in this subgroup the risk for events was uniformly therapy progressed to the end point compared with 28 (4.6%) low, independent of the extent of albuminuria at baseline. receiving non-ACEI therapy (P=0.49). During the extension The above findings were not explained by differences in study, however, 57 patients receiving ACEI therapy progressed patient characteristics or concomitant treatments because

1720 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 1717–1724, 2012 www.jasn.org CLINICAL EPIDEMIOLOGY

relationship between albuminuria and events among patients with left ventricular hypertrophy. The Framingham Heart Study showed that 6-year risk for cardiovascular disease was three-fold higher in nonhypertensive, nondiabetic persons with a urinary albumin-to-creatinine ratio above the sex-specific median (3.9 mg/min for men and 7.5 mg/min for women) than in those with a ratio below that level.16 Consistently, the Prevention of Renal and Vascular End Stage Disease (PREVEND) study found that independent of the effects of other cardiovascular risk factors, UAE was a predictor of cardiovascular mortality in the general population; the excess cardiovascular risk was already apparent at levels currently considered to be normal.7 All the preceding studies, however, included patients with a UAE ranging from Figure 1. Adjusted HRs for major cardiovascular events according to baseline albu- very low levels, in the so-called normoal- minuria. Solid line shows estimated relation when logarithmic hazard is modeled as buminuric range, to the micro- and even m linear function of log (UAE). The reference value (HR = 1) is set at UAE = 1 g/min. The macroalbuminuric ranges. Thus, those re- shaded area includes the 95% CIs for more general functional relation, as estimated by sults were largely driven by the well known P-splines. The HRs are adjusted for age, sex, duration of diabetes, history of cardio- association between micro- or macroalbu- vascular events, smoking habit, body mass index, mean BP, logarithms of HbA1c, LDL- to-HDL cholesterol ratio, triglyceride and serum creatinine levels, and whether patient minuria and excess cardiovascular risk. was allocated to ACEI therapy. CVD, cardiovascular disease. Conversely, the present study allows us to conclude that, as for BP, the concept of a threshold level to define normality is in- consistent with available data17 and that demographic, clinical, and laboratory variables at baseline, as any degree of measurable albuminuria—that is, a urinary al- well as targets for antihypertensive, antidiabetic, and lipid- bumin concentration close to the detection limit (1–2 mg/ml) lowering therapy throughout the whole observation period, of the methods commonly used to measure urinary albumin were similar between treatment groups. Thus, the finding that (such as nephelometry or immunoturbidimetry)—bears a patients originally randomly assigned to trandolapril during significant risk for cardiovascular events. BENEDICT tended to have fewer cardiovascular events than The preceding findings may have practical implications those who received the same treatment only after inclusion in because albuminuria can be reduced by amelioration of insulin the extension study was consistent with the hypothesis that sensitivity,18 weight loss,19 BP, and blood glucose reduction20 early ACEI therapy is more cardioprotective than late inte- and renin-angiotensin system inhibitor therapy.9,21 In the rvention. These data—which require confirmation in ad hoc long run, these interventions are expected to prevent or delay prospective studies—extend previous evidence of sustained end-organ damage.22 However, whether the above interven- long-term benefit of early intensified metabolic control in an tions are uniformly beneficial throughout the whole range of extension study of the Diabetes Control and Complications normoalbuminuria (or, rather, whether there is a threshold Trial 12 and suggest that the same “memory” effect could apply below which specific intervention is not associated with ap- also to ACEI, as already observed in the Heart Outcomes Pre- preciable benefits) is unknown. vention Evaluation (HOPE) study extension.13 In this regard, it is noteworthy that in this study, the A qualifying aspect of the present study was that for the first relationship between albuminuria and cardiovascular events time evidence for a continuous relationship between albu- was not appreciable in patients allocated to ACEI therapy minuria and risk was formally provided in patients with during BENEDICT. Actually, in this treatment group baseline normoalbuminuria to start with. The HOPE study14 found albuminuria was no longer predictive of cardiovascular events that in patients at increased cardiovascular risk, the relation- throughout the whole observation period. This was probably ship between albuminuria and cardiovascular events extended explained by a trend toward a progressively larger cardiopro- to as low as 0.5 mg of urinary albumin for 1 mmol of urinary tective effect of trandolapril for progressively increasing levels creatinine, while the Losartan Intervention For End-point re- of albuminuria. Independent of the above, during the exten- duction in hypertension (LIFE) study15 found a similar sion study we observed fewer events in patients who had been

J Am Soc Nephrol 23: 1717–1724, 2012 Albuminuria and Cardiovascular Disease 1721 CLINICAL EPIDEMIOLOGY www.jasn.org

Authorities Registry. Moreover, baseline characteristics of patients with or without extension data were similar, as well as their original allocation to ACE or non-ACEI therapy. A major strength is the centralized measurement of albuminuria—along with all other laboratory variables—by gold standard procedures in triplicate overnight urine collections.9,10,24 Study findings may be widely generalizable because outcome data were observed in patients with type 2 diabetes with normoalbuminuria and hypertension, a type of patient represent- ing at least 90% of the whole diabetic population. In addition, the follow-up was the longest among similar studies thus far reported that considered the relationship between baseline albuminuria and cardiovascular outcomes in patients with or without diabetes. Finally, consistency of the results obtained by using fractional Figure 2. Adjusted HRs for major cardiovascular events according to baseline albu- polynomial models or spline functions minuria in patients who during the BENEDICT trial had been randomly allocated to provided additional evidence of the robust- fi ACEI therapy with trandolapril alone or combined to verapamil (solid line) or to non- ness of the study ndings. Another unique ACEI therapy with verapamil alone or placebo (dashed line). The lines show estimated strength was that, unlike data from previ- relation when logarithmic hazard is modeled as linear function of log (UAE). The ref- ous series in the general population7,16 or erence value (HR = 1) is set at UAE = 1 mg/min. The HRs are adjusted for age, sex, in patients with left ventricular hypertro- duration of diabetes, history of cardiovascular events, smoking habit, body mass in- phy15 or increased cardiovascular risk14 dex, mean BP, logarithms of HbA1c, LDL-to-HDL cholesterol ratio, triglyceride and that included also patients with micro- or serum creatinine levels, and whether patient was allocated to ACEI therapy. CVD, macroalbuminuria (who most likely cardiovascular disease. “drove” the study findings), our present data definitely confirmed the independent predictive value of albuminuria in a pure originally randomly assigned to ACEI therapy at inclusion in population of patients with type 2 diabetes and normoalbu- BENEDICT than in those who started ACEI therapy only minuria. Whether the above findings can be generalized to afterconclusion of the trial. These findings extendpreviousand nonwhite populations or to persons without diabetes is matter more robust data from HOPE–The Ongoing Outcomes study of investigation. showing that the protective effect of ACEI therapy against In conclusion, in patients with type 2 diabetes and cardiovascular events observed during the HOPE trial was normoalbuminuria,anydegree of measurable urinary albumin sustained during 2.6 years extended follow-up, while all pa- bears a significant risk for cardiovascular events. The associ- tients were receiving the same ACEI therapy.13 These findings ation between albuminuria and risk is continuous, and there led the authors to suggest that the 4.5 years of initial “earlier” is no threshold level that distinguishes patients at risk from use of ramipril therapy during the HOPE trial provided those who are protected from cardiovascular disease. This greater cardioprotection compared with later initiation,13 incremental risk almost fully dissipates with early ACEI possibly because of benefits on endothelial or vascular struc- therapy. Future randomized trials are needed to identify levels ture and function that persisted beyond the blinded treatment of albuminuria above which cardioprotective therapy is period of the trial.23 Conceivably, the above considerations appreciably beneficial in this population. could apply also to our study population. The major limitation of this study is that this was an extension of a clinical trial originally designed for other purposes. CONCISE METHODS Thus, study findings are hypothesis generating and need to be tested in ad hoc prospective studies. However, extension data The BENEDICT Extension Study could be recorded on the basis of preplanned monitoring BENEDICT-A has been described elsewhere.9,10,24 In brief, from guidelines in almost 90% of patients completing BENEDICT, 1997 to 2000, a total of 1209 patients with type 2 diabetes and and exhaustive survival data could be obtained by the Health hypertension, normoalbuminuria, creatinine ,1.5 mg/dl, and

1722 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 1717–1724, 2012 www.jasn.org CLINICAL EPIDEMIOLOGY

HbA1c ,11% were randomly assigned to trandolapril, verapamil, between continuous variables and outcomes.25,26 In preplanned sen- their combination, or placebo in order to prevent microalbuminuria. sitivity analyses, multivariable models were restricted to low-risk pa- The current study was a preplanned extension study of BENEDICT-A tients: those without previous cardiovascular events (n=1156) and that consisted of clinical, laboratory, and case record evaluations until those persistently normoalbuminuric throughout BENEDICT December 31, 2008. Finally, the Registry of the Health District pro- (n=1107). Finally, analyses separately considered data from BENE- vided fatal events and causes of deaths. On the basis of BENEDICT DICTand the extension study and from patients originally randomly results, after the trial (January 2004), all patients received an ACEI. assigned to ACEI therapy or not. Data were analyzed using SPSS Overall, outcome data from BENEDICT with or without extension software, version 17.0.1, for Windows (SPSS, Inc., Chicago, IL) and data were available for 1056 of the 1208 patients (87.4%) (Supplemen- Stata software, version 11.0 (fracpoly command; Stata Corp., Cary, tal Figure 1). NC).

Definitions and Measurements Baseline normoalbuminuria was defined as UAE ,20 mg/min for two ACKNOWLEDGMENTS of three overnight urine collections, and the median of the three measurements was used for analysis. Progression to microalbumin- The authors are indebted to the staff of the Diabetology Units and of uria was diagnosed in patients with UAE $20 mg/min and ,200 mg/ the Clinical Research Center for Rare Diseases “Aldo & Cele Daccò” of min in two of three measurements. the Mario Negri Institute for their assistance in the selection of and care for the patients in this study; to Diletta Valsecchi for monitoring Cardiovascular Outcomes the data handling; and to Manuela Passera for assistance with the The main outcome was the first onset of a component of a com- preparation of the manuscript. posite end point of fatal (including sudden death) or nonfatal major Abbott (Ludwigshafen, Germany) sponsored BENEDICT. E.P. is cardiovascular events, such as coronary (acute myocardial infarction, the recipient of a long-term fellowship from the European Renal unstable angina pectoris, or coronary revascularization by bypass Association–European Dialysis Transplant Association, which al- grafting or percutaneous angioplasty), cerebrovascular (stroke, tran- lowed him a postdoctoral position in the Mario Negri Institute. sient ischemic attack, pre–cerebral artery revascularization), or pe- This study was an academic, internally funded study. Nosponsoror ripheral vascular (amputation, revascularization) disease and company was involved in the data recording; study design, analysis, hospitalizations due to congestive heart failure. All events were defined interpretation, and reporting; manuscript preparation; or decision to a priori and were adjudicated under blind conditions by P.R. and E.P. submit the article for publication. For a complete list of study in- Secondary outcomes were spontaneous components of the com- vestigators, see the Supplemental Material. posite end point and coronary events.

Statistical Analyses The relationships between baseline albuminuria and cardiovascular DISCLOSURES events were evaluated by Cox proportional hazards models. Con- None. founders included baseline variables with a proven or possible association with the outcome, such as age, sex, duration of diabetes, smoking, history of cardiovascular events, body mass index, serum REFERENCES creatinine, HbA1c levels, randomization to ACEI therapy during the core trial, mean arterial pressure, LDL-to-HDL cholesterol ratio, tri- 1. Adler AI, Stevens RJ, Manley SE, Bilous RW, Cull CA, Holman RR; glyceride and uric acid levels, and treatment with lipid-lowering UKPDS GROUP: Development and progression of nephropathy in type therapy. Continuous variables, including albuminuria, were kept 2 diabetes: The United Kingdom Prospective Diabetes Study (UKPDS – continuous. Colinearity was tested by Pearson correlation coefficient; 64). Kidney Int 63: 225 232, 2003 2. Dinneen SF, Gerstein HC: The association of microalbuminuria and when colinearity was found, only one variable (HbA instead of 1c mortality in non-insulin-dependent diabetes mellitus. A systematic glucose, LDL cholesterol instead of total cholesterol) or a surrogate overview of the literature. Arch Intern Med 157: 1413–1418, 1997 (mean for systolic or diastolic BP and the LDL-to-HDL ratio for 3. Nelson RG, Pettitt DJ, Carraher MJ, Baird HR, Knowler WC: Effect of both components of the ratio) was used. Variables with a nonlinear proteinuria on mortality in NIDDM. Diabetes 37: 1499–1504, 1988 4. Rayner M, Petersen S, Buckley C, Press V: Coronary heart disease statistics: association with risk (LDL-to-HDL ratio, albuminuria, and HbA1c Diabetes supplement. 2001 edition. Available at: http://www.bhf.org.uk/ and creatinine levels) were log-transformed. The proportionality publications/view-publication.aspx?ps=1001401. Accessed on August 8, assumption was assessed using the log-rank and the weighted 2012 Schoenfeld residuals, and model goodness of fit was assessed by the 5. Mattock MB, Barnes DJ, Viberti G, Keen H, Burt D, Hughes JM, Hosmer-Lemeshow test. Fitzgerald AP, Sandhu B, Jackson PG: Microalbuminuria and coronary – Multivariable analysis was also performed by modeling baseline heart disease in NIDDM: An incidence study. Diabetes 47: 1786 1792, 1998 albuminuria with the fractional polynomial algorithm method (first 6. Borch-Johnsen K, Feldt-Rasmussen B, Strandgaard S, Schroll M, Jensen fl and second degree), or a spline function, two exible and informative JS: Urinary albumin excretion. An independent predictor of ischemic approaches for the evaluation of possible nonlinear relationships heart disease. Arterioscler Thromb Vasc Biol 19: 1992–1997, 1999

J Am Soc Nephrol 23: 1717–1724, 2012 Albuminuria and Cardiovascular Disease 1723 CLINICAL EPIDEMIOLOGY www.jasn.org

7. Hillege HL, Fidler V, Diercks GF, van Gilst WH, de Zeeuw D, van individuals: The Framingham Heart Study. Circulation 112: 969–975, Veldhuisen DJ, Gans RO, Janssen WM, Grobbee DE, de Jong PE; 2005 Prevention of Renal and Vascular End Stage Disease (PREVEND) Study 17. Forman JP, Brenner BM: ‘Hypertension’ and ‘microalbuminuria’:The Group: Urinary albumin excretion predicts cardiovascular and non- bell tolls for thee. Kidney Int 69: 22–28, 2006 cardiovascular mortality in general population. Circulation 106: 1777– 18. Pistrosch F, Herbrig K, Kindel B, Passauer J, Fischer S, Gross P: 1782, 2002 Rosiglitazone improves glomerular hyperﬁltration, renal endothelial 8. Ruggenenti P, Remuzzi G: Time to abandon microalbuminuria? Kidney dysfunction, and microalbuminuria of incipient diabetic nephropathy in Int 70: 1214–1222, 2006 patients. Diabetes 54: 2206 –2211, 2005 9. Ruggenenti P, Fassi A, Ilieva AP, Bruno S, Iliev IP, Brusegan V, Rubis N, 19. Chagnac A, Weinstein T, Herman M, Hirsh J, Gafter U, Ori Y: The effects Gherardi G, Arnoldi F, Ganeva M, Ene-Iordache B, Gaspari F, Perna A, of weight loss on renal function in patients with severe obesity. JAm Bossi A, Trevisan R, Dodesini AR, Remuzzi G; Bergamo Nephrologic Di- Soc Nephrol 14: 1480–1486, 2003 abetes Complications Trial (BENEDICT) Investigators: Preventing micro- 20. UK Prospective Diabetes Study Group: Tight blood pressure control albuminuria in type 2 diabetes. N Engl J Med 351: 1941–1951, 2004 and risk of macrovascular and microvascular complications in type 2 10. Ruggenenti P, Perna A, Ganeva M, Ene-Iordache B, Remuzzi G; BEN- diabetes: UKPDS 38. BMJ 317: 703–713, 1998 EDICT Study Group: Impact of blood pressure control and angiotensin- 21. Parving HH, Lehnert H, Bröchner-Mortensen J, Gomis R, Andersen S, converting enzyme inhibitor therapy on new-onset microalbuminuria in Arner P; Irbesartan in Patients with Type 2 Diabetes and Micro- type 2 diabetes: A post hoc analysis of the BENEDICT trial. JAmSoc albuminuria Study Group: The effect of irbesartan on the development Nephrol 17: 3472–3481, 2006 of diabetic nephropathy in patients with type 2 diabetes. N Engl J Med 11. De Cosmo S, Motterlini N, Prudente S, Pellegrini F, Trevisan R, Bossi A, 345: 870–878, 2001 Remuzzi G, Trischitta V, Ruggenenti P; BENEDICT Study Group: Im- 22. Brouwers FP, Asselbergs FW, Hillege HL, de Boer RA, Gansevoort RT, pact of the PPAR-gamma2 Pro12Ala polymorphism and ACE inhibitor van Veldhuisen DJ, van Gilst WH: Long-term effects of fosinopril and therapy on new-onset microalbuminuria in type 2 diabetes: evidence pravastatin on cardiovascular events in subjects with microalbuminuria: from BENEDICT. Diabetes 58: 2920–2929, 2009 Ten years of follow-up of Prevention of Renal and Vascular End-stage 12. Writing Team for the Diabetes Control and Complications Trial/Epi- Disease Intervention Trial (PREVEND IT). Am Heart J 161: 1171–1178, demiology of Diabetes Interventions and Complications Research Group: 2011 Sustained effect of intensive treatment of type 1 diabetes mellitus on 23. Mancini GB, Henry GC, Macaya C, O’Neill BJ, Pucillo AL, Carere RG, development and progression of diabetic nephropathy: the Epidemiology Wargovich TJ, Mudra H, Lüscher TF, Klibaner MI, Haber HE, Uprichard of Diabetes Interventions and Complications (EDIC) study. JAMA 290: AC, Pepine CJ, Pitt B: Angiotensin-converting enzyme inhibition with 2159–2167, 2003 quinapril improves endothelial vasomotor dysfunction in patients with 13. Bosch J, Lonn E, Pogue J, Arnold JM, Dagenais GR, Yusuf S; HOPE/ coronary artery disease. The TREND (Trial on Reversing ENdothelial HOPE-TOO Study Investigators: Long-term effects of ramipril on car- Dysfunction) Study. Circulation 94: 258–265, 1996 diovascular events and on diabetes: Results of the HOPE study ex- 24. BENEDICT Group: The BErgamo NEphrologic DIabetes Complications tension. Circulation 112: 1339–1346, 2005 Trial (BENEDICT): Design and baseline characteristics. Control Clin 14. Gerstein HC, Mann JF, Yi Q, Zinman B, Dinneen SF, Hoogwerf B, Hallé JP, Trials 24: 442–461, 2003 Young J, Rashkow A, Joyce C, Nawaz S, Yusuf S; HOPE Study Inves- 25. Royston P, Altman DG, Sauerbrei W: Dichotomizing continuous pre- tigators: Albuminuria and risk of cardiovascular events, death, and heart dictors in multiple regression: A bad idea. Stat Med 25: 127–141, 2006 failure in diabetic and nondiabetic individuals. JAMA 286: 421–426, 2001 26. Royston P, Sauerbrei W: Multivariable Model-Building. A Pragmatic 15. Wachtell K, Ibsen H, Olsen MH, Borch-Johnsen K, Lindholm LH, Approach to Regression Analysis Based on Fractional Polynomials for Mogensen CE, Dahlöf B, Devereux RB, Beevers G, de Faire U, Fyhrquist Modelling Continuous Variables, West Sussex, United Kingdom, J F, Julius S, Kjeldsen SE, Kristianson K, Lederballe-Pedersen O, Wiley, 2008. Nieminen MS, Okin PM, Omvik P, Oparil S, Wedel H, Snapinn SM, Aurup P: Albuminuria and cardiovascular risk in hypertensive patients with left ventricular hypertrophy: The LIFE study. Ann Intern Med 139: See related editorial, “Urinary Albumin: How Low Is Normal?,” on pages 1605– – 901 906, 2003 1607. 16. Arnlöv J, Evans JC, Meigs JB, Wang TJ, Fox CS, Levy D, Benjamin EJ, D’Agostino RB, Vasan RS: Low-grade albuminuria and incidence of This article contains supplemental material online at http://jasn.asnjournals. cardiovascular disease events in nonhypertensive and nondiabetic org/lookup/suppl/doi:10.1681/ASN.2012030252/-/DCSupplemental.

1724 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 1717–1724, 2012 COMPLETE METHODS

The BENEDICT Extension Study

Between February 1997 and July 2000, 1209 consenting type 2 diabetes patients with arterial hypertension, normoalbuminuria, serum creatinine levels <1.5 mg/dl and HbA1c

<11%, were randomized into the BENEDICT study, a randomized, double blind, controlled clinical trial. The study was run by the Clinical Research Center for Rare

Diseases “Aldo & Cele Daccò” of the Mario Negri Institute for Pharmacological

Research in co-operation with nine Diabetology Units (all in Italy) and aimed to compare the protective effect of at least 3 year treatment with the ACE inhibitor

Trandolapril (2 mg/day), the ndCCB Verapamil (180 mg/day), their combination, or placebo against the development of microalbuminuria. in this population (eFigure 1,

Online-Only). The rationale, design and results of this study have been reported in detail elsewhere9-11. Since January 2004, When the BENEDICT Trial was closed, by January

2004, study participants continued to be monitored until December 31, 2008 by preplanned serial clinical and laboratory evaluations and dedicated case record forms at the

Outpatient Clinics of the Clinical Research Center and of eight of the nine Diabetology

Units originally involved in the Trial. Based on BENEDICT results all of them were maintained on ACE inhibitors therapy independent of their original randomization in the trial. For all patients, complete information on deaths including fatal cardiovascular events and causes of deaths were obtained from the Registry of the Health District

(Azienda Sanitaria Locale) of the Bergamo Province. Of the 1208 participants originally randomized in BENEDICT, 1195 were still alive at the end of the trial. For 139 of these patients, only outcome data recorded during the trial were available since 33 of had been included by the Unit of Diabetology of Cagliari that for technical reasons was were not involved in the present Extension study for technical reasons and 104 did not consent to enter the Extension study or were lost to follow-up (eFigure 1, Online-Only).

Overall, outcome data from BENEDICT trial with or without additional Extension data were available for 1056 of the 1208 patients (87.4%) originally randomized in

BENEDICT. The study protocol was approved by the institutional review boards of the

Clinical Research Center and of involved Diabetology Units. Participants provided written informed consent to study inclusion.

Definitions and measurements

Type 2 diabetes was diagnosed according to WHO criteria. Arterial Hypertension was defined as a systolic blood pressure (BP) >130 mmHg and/or a diastolic BP >85 mmHg, or concomitant BP lowering therapy, and normoalbuminuria as a urinary albumin excretion rate <20 !g/min in at least two of three consecutive timed overnight urine collections at inclusion. The median value of the three baseline albuminuria measurements was used for analysis. Progression to persistent microalbuminuria

(primary outcome variable of the BENEDICT Trial) was diagnosed in patients with a urinary albumin excretion rate > 20 !g/min and < 200 !g/min in at least two of three consecutive measurements in two consecutive visits. Throughout the whole observation period of the BENEDICT trial and the subsequent BENEDICT Extension Study

Albuminuria was centrally measured at the Laboratories of the Clinical Research Center by nephelometry (Beckman Array System). All the laboratory parameters considered during the BENEDICT trial and on subsequent follow-up were also centrally measured at the same Laboratories by means of a Beckman Syncron Cx5 instrument and a Coulter

Maxm (Beckman Coulter).

Cardiovascular Outcomes

The main outcome variable of the BENEDICT Extension Study was the first onset of one of the components of a composite end-point of fatal (including sudden death) or non-fatal major cardiovascular events such as events related to coronary (acute myocardial infarction, unstable angina pectoris or coronary revascularization by bypass grafting or percutaneous transluminal angioplasty), cerebrovascular (stroke, transient ischemic attack, pre-cerebral artery revascularization) or peripheral vascular

(amputation, revascularization) disease and hospitalizations because of congestive heart failure. Secondary outcomes were: i. the first onset of one of the components of the composite endpoint without

considering treatment interventions such as revascularizations and amputations ii. the first onset of a coronary event considered separately from other cardiovascular

events.

All the events were defined a priori and were adjudicated by a cardiologist (P.R.) and an epidemiologist (E.P.), who were blinded to patient treatment allocation.

Statistical Analysis

Continuous Characteristics of participants with or without cardiovascular events were compared by unpaired t test or Mann-Whitney or Kruskal-Wallis test, categorical or dicothomous characteristics by Pearson’s "2 or Fisher’s exact test as appropriate.

The primary analysis was based on 1208 normoalbuminuric patients at randomization.

The relationships between baseline albuminuria and cardiovascular events observed during the BENEDICT trial or the Extension study, were evaluated by using Cox proportional hazards regression models. To account for potential Confounders, multivariable models included baseline variables that a priori had been considered to have a proven or possible association with the outcome, such as age, gender, duration of diabetes, smoking habits, history of cardiovascular events, body mass index, serum creatinine, Hba1C levels, randomization to ACE-inhibitor therapy Yes or No during the

Core Trial, mean arterial pressure (MAP), low-density-lipoprotein (LDL)/high-density- lipoprotein (HDL) cholesterol ratio, triglycerides and uric acid levels, and concomitant treatment with lipid lowering therapy. The association of the remaining baseline covariates with considered outcomes was explored at univariate analyses. Since no significant association was observed, no other variable was considered in the multivariable models. All continuous variables, including albuminuria, were kept continuous for the analyses. Co-linearity between baseline covariates was tested by the

Pearson correlation coefficient and when a co-linearity between two variables was found, only one of the two variables (HbA1C instead of blood glucose, LDL cholesterol instead of total cholesterol) or a surrogate of both variables (MAP for both systolic and diastolic BP its components and the LDL/HDL cholesterol ratio for LDL and HDL both cholesterols considered separately) variable were used.

Comparisons according to randomization to ACE –inhibitor (yes or no) (y/n) were performed using the intention-to-treat principle. Departure from linearity of risk was tested creating multiple categorical variables and those with a non-linear gradient when founded (LDL/HDL cholesterol ratio, albuminuria, HbA1C and creatinine levels) were log-transformed.

Variables with a non-linear association with risk – tested by multiple categorical variables- were log-transformed (LDL/HDL ratio, albuminuria, HbA1C and creatinine levels).

The proportionality hazard assumption was assessed using the long-rank and the weighted Schoenfeld residuals, and the fit goodness of the model by the Hosmer-

Lemeshow test. Multivariable survival analysis was also performed by modelling baseline albuminuria withthe fractional polynomial algorithm method (first and second degree), or a spline function,two flexible and informative approaches for the evaluation of possible nonlinear relationships between continuous variables and outcomes.12,13

Sensitivity analyses were pre-planned to test the study findings in specific contexts.

The study findings were also tested in pre-planned sensitivity analyses. Thus, multivariable analyses models were also restricted to (a) low-risk subjects: those without previous history of major cardiovascular events (n=1156), or to those who had been persistently normoalbuminuric throughout the BENEDICT trial (n=1107), (b) specific outcomes: Sensitivity analyses also considered those with spontaneous cardiovascular events independent of (excluding revascularizations and amputations), or those only with coronary events independent of other cardiovascular events. Finally, the relationships between baseline covariates and outcomes the principal analysis were also evaluated separately within the BENEDICT trial and Extension study observation periods and within subjects originally randomized to ACE inhibitor therapy yes or not.

Data were expressed as n (%), mean and Standard Deviation (SD) or Median and interquartile range (IQR) as appropriate and a p value < 0.05 was considered as statistically significant. Data were analysed using SPSS 17.0.1 for Windows (Chicago,

IL) and STATA 11.0 (fracpoly command). ! 10-YEAR CARDIOVASCULAR RISK IN TYPE 2 DIABETES IS PREDICTED BY MEASURABLE

URINARY ALBUMIN EVEN IN THE NORMOALBUMINURIC RANGE:

THE BENEDICT EXTENSION STUDY

ONLINE-ONLY MATERIAL

eTable 1:

Baseline characteristics of patients with and without complete follow-up.

eTable2:

Hazard Ratios (95% Confidence Intervals) for the primary end point (first onset of fatal or non-fatal major cardiovascular events) for each 1 µg/min range in baseline urinary albumin excretion (UAE) compared to UAE < 1 µg/min (reference value) in the study group as a whole (Overall) or according to randomization to ACE inhibitor therapy Yes or No.

eFigure 1:

Study Flow-Chart. eTable 1: Baseline characteristics of patients with and without complete follow-up.

Overall Core + Core p§ extension only N (%) N= 1208 N= 1069 (88.5) N= 139 (11.5)

Age – yr 62.33 ± 8.05 62.29 ± 7.940 62.64± 8.88 0.76 Male sex – no. (%) 638 (52.8) 584 (54.6) 54 (38.8) < 0.001 History of CV events 52 (4.3) 47 (4.4) 5 (3.6) 0.43 Know duration of diabetes – yr 6 (3-11) 6 (2-11) 10 (3-16) < 0.001 Smoking status – no. (%) Never smoked 700 (57.9) 608 (56.9) 92 (66.2) Former smoker 362 (30.0) 328 (30.7) 34 (24.5) 0.11 Current smoker 146 (12.1) 133 (12.4) 13 (9.4) Body Mass Index † 29.08 ± 4.72 29.06± 4.72 29.20± 4.69 0.75 Trough blood pressure – mmHg Systolic 150.82 ±14.16 150.90 ± 14.11 150.22 ± 14.57 0.60 Diastolic 87.46 ± 7.63 87.52 ± 7.54 87.04 ± 8.31 0.51 Mean 108.58 ± 8.35 108.64 ± 8.27 108.10 ± 8.94 0.49 Glycosilated hemoglobin - % 5.79 ± 1.37 5.76 ± 1.36 6.10 ± 1.463 0.014 Glucose – mg/dL 161.34 ± 47.05 160.25 ± 46.09 170.55 ± 53.90 0.020 Cholesterol - mg/dL Total 209.78 ± 36.74 208.82 ± 36.67 217.94 ± 36.393 0.009 High-density lipoprotein 46.94 ± 12.06 46.5759 ± 11.760 50.0476 ± 14.040 0.002 Low-density lipoprotein 162.75 ± 36.05 162.18 ± 36.08 167.53 ± 35.54 0.11 Triglycerides - mg/dL 125 (91-181) 125 (91-182) 130.5 (95-178) 0.74 Serum creatinine - mg/dL 0.90 ± 0.16 0.91 ± 0.16 0.88 ± 0.17 0.069 Uric acid - mg/dL 4.09 ± 1.18 5.03 ± 1.18 4.80 ± 1.24 0.057 Albuminuria - µg/min 5.24 (3.5-9.3) 5.26 (3.6-9.4) 4.99 (3.4-9.0) 0.33 Medications Allocation to the study treatments Placebo 302 (25.0) 270 (25.3) 32 (23.0) Verapamil 303 (25.1) 270 (25.3) 33 (23.7) 0.87 Trandolapril 302 (25.0) 265 (24.8) 37 (26.6) Verapamil + Trandolapril 301 (24.9) 264 (24.7) 37 (26.6) Antihypertensive agents – no (%)* 664 (55.0) 579 (54.2) 85 (62.0) 0.05 Total Number of antihypertensive agents** 2 (1-2) 2 (1-3) 2 (1-3) 0.23 Antilipidemic agents – no (%) 138 (11.4) 117 (10.9) 21 (15.3) 0.09 Aspirin – no (%) 28 (2.3) 25 (2.3) 3 (2.2) 0.60

* Different from the study treatment. **Includes study treatment and other antihypertensive drugs. §: comparisons between patients with and without complete follow-up.

eTable2:

Hazard Ratios (95% Confidence Intervals) for the primary end point (first onset of fatal or non- fatal major cardiovascular events) for each 1 µg/min range in baseline urinary albumin excretion

(UAE) compared to UAE < 1 µg/min (reference value) in the study group as a whole (Overall) or according to randomization to ACE inhibitor therapy Yes or No. In the study group as a whole and in patients not randomized to ACEi therapy, each 1 µg/min UAE range is associated with a significant excess cardiovascular risk compared to the reference value. In patients originally randomized to ACEi therapy, the risk of events associated with each UAE range is never significantly different from that associated with the reference value. Albuminuria is modeled with the second degree fractional polynomial transformation. Multivariable Cox proportional hazards regression analyses adjusted for the same variable shown in Table 3.

UAE OVERALL ACEi No ACEI Yes (!g/min) Ranges HR 95% CI HR 95% CI HR 95% CI < 1 1.00 -- 1.00 -- 1.00 -- 1-2 1.04 1.02-1.07 1.02 1.01-1.04 0.95 0.55-1.61 2-3 1.10 1.04-1.18 1.08 1.04-1.12 0.95 0.42-2.11 3-4 1.20 1.08-1.33 1.17 1.08-1.26 0.97 0.37-2.55 4-5 1.31 1.12-1.54 1.30 1.14-1.48 0.99 0.34-2.93 5-6 1.45 1.16-1.80 1.47 1.20-1.80 1.03 0.33-3.25 6-7 1.61 1.21-2.13 1.70 1.30-2.22 1.08 0.33-3.54 7-8 1.78 1.28-2.47 1.97 1.40-2.76 1.13 0.34-3.80 8-9 1.96 1.34-2.86 2.28 1.51-3.43 1.18 0.35-3.99 9-10 2.14 1.40-3.25 2.65 1.64-4.30 1.25 0.37-4.18 10-11 2.30 1.46-3.60 2.90 1.71-4.90 1.31 0.39-4.34 11-12 2.45 1.52-3.89 3.11 1.77-5.44 1.38 0.42-4.52 12-13 2.49 1.56-4.00 3.15 1.76-5.63 1.45 0.45-4.65 13-14 2.49 1.55-3.99 3.00 1.67-5.40 1.53 0.49-4.80

1209 participants randomized in the BENEDICTTrial trial

604 assigned to ACE inhibitor therapy with 605 assigned to non-ACE inhibitor therapy Trandolparil alone or in combination with with Verapamil alone or placebo verapamil

1 excluded due to microalbuminuria at inclusion

603 treated and followed in BENEDICT 605 treated and followed in BENEDICT 6 deaths (1 from major CV event) 7 deaths (3 from major CV events) 26 non-fatal major CV events 25 non-fatal major CV events 8 lost to follow up 8 lost to follow up

597 eligible for the Extension Study 598 eligible for the Extension Study

74 without 65 without Extension data Extension data

523 with Extension Follow-up data 533 with Extension Follow-up data 81 deaths (17from major CV events: 11 at 74 deaths (16 from major CV events: 9 at first event, 6 at second or subsequent first event, 7at second or subsequent events)) events)) 46 non-fatal major CV events 68 non-fatal major CV events

603 available for primary analyses 605 available for primary analyses

eFigure STUDY ORGANIZATION

Members of the BENEDICT Study Organization were as follows (all in Italy unless otherwise noted): Principal investigator — G. Remuzzi (Bergamo); Study coordinator

— P. Ruggenenti (Bergamo); Coordinating center — Mario Negri Institute for

Pharmacological Research, Clinical Research Center for Rare Diseases Aldo e Cele

Daccò, Villa Camozzi, Ranica (Bergamo); Participating centers — G. Nastasi, A.

Ongaro, F. Querci, A. Anabaya (Alzano Lombardo); R. Trevisan, A.R. Dodesini, G.

Lepore, I. Nosari, C. A. Aros Espinoza, A. Fassi (Bergamo); M. Songini, G. Carta, G.

Piras (Cagliari); B. Minetti, P. Fiorina, G. Ghilardi, V. Grassia, E. Pezzali, E. Seghezzi,

I. Villanova (Clusone); A. Spalluzzi, I. Codreanu, C. Flores (Ponte San Pietro, Villa d'Almè); C. Chiurchiu, F. Arnoldi, L. Mosconi, M. Monducci (Ranica); A. Bossi, M.

Facchetti, V. Brusegan (Romano di Lombardia); F. Inversi, V. Bertone, R. Mangili, S.

Bruno (Seriate); A. Bossi, A. Parvanova, I.P. Iliev, E. Terzieva (Treviglio);

Ophthalmologists — M. Filipponi, I.P. Iliev, S. Tadini (Bergamo); Monitoring and drug distribution (Mario Negri Institute) — G. Gherardi, N. Rubis, S. Birolini, L. Bruni, W.

Calini, V.A. Carrasco Oyarzun, R. D’Adda, O. Diadei, M. Ferrari, L. Mangili, A.

Milani, G. Noris, K. Pagani, S. Quadri, A. Rossi, S. Secomandi, G. Villa (Ranica);

Carriers (Mario Negri Institute) — G. Gaspari, S. Gelmi, G. Gervasoni, L. Nembrini

(Ranica); Database and data validation (Mario Negri Institute ) — A. Remuzzi, B. Ene-

Iordache, V. Gambara (Ranica); Data analysis (Mario Negri Institute) — A. Perna, B.D.

Dimitrov, M. Ganeva, J. Zamora (Ranica); Laboratory measurements (Mario Negri

Institute) — F. Gaspari, F. Carrara, E. Centemeri, S. Ferrari, M. Pellegrino, N. Stucchi

(Ranica); Genomic evaluations (Mario Negri Institute) — M. Noris, P. Bettinaglio, S.

Bucchioni, J. Caprioli, B. Giussani (Bergamo); Regulatory affairs (Mario Negri

Institute) — P. Boccardo (Ranica); Steering committee — S. Kupfer (Abbott Park, Ill., United States), L. Minetti (Bergamo), G. Remuzzi (Bergamo), U.F. Legler

(Ludwigshafen, Germany), B. Kalsch (Ludwigshafen, Germany), D. Nehrdich

(Ludwigshafen, Germany), A. Nicolucci (S. Maria Imbaro), A. Perna (Bergamo), P.

Ruggenenti (Bergamo); Safety committee — G.L. Bakris (Chicago, United States), R.

Kay (Sheffield, United Kingdom), G.C. Viberti (London, United Kingdom).