Available data suggest that effective antihypertensive treatment is the best inhibitor of diabetic nephropathy [7-10]. Angiotensin-converting enzyme inhibitors have been found more effective than placebo and ß-adrenergic blocking agents in hypertensive as well as in normotensive diabetics with early and advanced nephropathy [11-15]. Some classes of calcium antagonists effectively decrease urinary protein excretion and may preserve renal function. However, analysis of several studies shows that, although the effects of angiotensin-converting enzyme inhibitors are consistent, those of calcium antagonists vary [16-18].

Short-term studies showed a clear antiproteinuric effect of captopril and of enalapril on the diabetic kidney, probably independent of the antihypertensive effect of these agents [14, 15, 19]. However, the outcome of long-term intervention and the possibility of a true alteration of the natural course of the disease were unknown. We did a relatively long-term, 5-year study of the effect of the angiotensin-converting enzyme inhibitor, enalapril, on the course of diabetic nephropathy in normotensive, type II diabetic patients with microalbuminuria and normal renal function. Our report describes a randomized, placebo-controlled, double-blind study on 94 diabetic patients.

Methods

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Patients

A total of 108 patients with type II diabetes mellitus, diagnosed according to World Health Organization criteria [20] who attended six clinics in the Tel Aviv area were recruited during 1986 and gave informed consent to participate in the study.

The inclusion criteria were as follows: age less than 50 years; duration of diabetes mellitus of less than 10 years with no evidence of systemic, renal, cardiac, or hepatic diseases; body mass index less than 27 kg/m²; normal blood pressure values on two consecutive examinations (systolic, 140 mm Hg; diastolic, 90 mm Hg; mean blood pressure < 107 mm Hg); serum creatinine, < 123 µmol/L (1.4 mg/dL); and microalbuminuria (urinary protein excretion of 30 to 300 mg/24 h) on two consecutive visits without evidence of urinary tract infection.

Initially, there were 49 men and 59 women, ages 34 to 49 years (mean age [±SD], 44 ± 4 years). The duration of diabetes was 0.5 to 9.1 years (mean duration [±SD], 6.7 ± 1.6 years). Sixteen patients received insulin, 43 were taking oral hypoglycemic agents, and 49 were using diet to control their diabetes.

Protocol

The protocol was approved by the hospital review board. After a 2-month pretreatment period, the patients were randomly allocated to receive either 10 mg enalapril (Teva Pharmaceutical Industries, Ltd., Petach Tikwa, Israel) daily or placebo in a double-blind manner. The placebo tablets were similar but not identical to enalapril. Randomization was done using a table of random numbers [21]. The follow-up period was terminated, for each patient, exactly 5 years after his or her randomization, and the data were submitted for evaluation.

The patients were seen by their family physicians approximately every 3 to 4 months. On these visits, fasting blood glucose, glycosylated hemoglobin, serum creatinine, serum electrolyte levels, and albuminuria in 24-hour urine samples were determined. Blood pressure was measured by mercury sphygmomanometers with the patients sitting after a 5-minute rest; the average of two determinations was recorded. The diastolic pressure was determined at Korotkoff phase V. If systolic blood pressure values of 145 mm Hg, or diastolic values of 95 mm Hg, were found on two consecutive occasions, treatment with long-acting nifedipine (Pressolate, Agis Industries Ltd., Yeruham, Israel) was initiated. Funduscopy was done yearly by an ophthalmologist, and the presence of diabetic retinopathy was recorded.

Measurements

Glucose and creatinine levels were determined by routine automated methods. Glycosylated hemoglobin levels were measured by affinity chromatography with a commercial kit (Isolab, Biochemical Methodology, Drower 4350, Akron, Ohio). The albumin excretion rate was measured on 24-hour urine samples by an automated immunoturbidimetric assay [22].

Sixteen to 20 fasting blood glucose determinations and 15 to 20 glycosylated hemoglobin values were available for each patient. For each patient, the correlation coefficients between fasting blood glucose and glycosylated hemoglobin levels were between 0.60 and 0.84. The mean blood pressure values were calculated for each patient (mean pressure defined as diastolic value plus one third of the pulse pressure). The reciprocal creatinine value (100/creatinine value) was calculated for each visit [23], and the decline in renal function was expressed as a percentage of the initial value. The course of renal function, of the mean blood pressure, and of urinary protein excretion were plotted against time (separately) for the enalapril and the placebo groups.

Statistical Analysis

All data were expressed as mean (±SD) and ranges. Significance was defined as P < 0.05. To test for adequate randomization, the enalapril and placebo groups were compared with respect to mean age, mean duration of diabetes, as well as mean baseline values of albuminuria, serum creatinine, glycosylated hemoglobin, and mean blood pressure using pooled variance Student t-tests for independent groups as well as one-way analysis of variance. To compare the annual means of the various measurements between the two groups and within each group, one between-group factor and one repeated measures factor were used in analysis of variance. The rate of decrease of reciprocal creatinine levels and the rate of increase of albuminuria were calculated by linear regression analysis.

Results

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Five patients, 2 taking enalapril and 3 taking placebo left the study during the first year. Six patients (4 taking enalapril and 2 taking placebo) developed a disturbing cough, and the treatment was discontinued. Three patients (1 on enalapril and 2 on placebo) were lost to follow-up during the third and fourth years. The final analysis was therefore done on 94 patients, of whom 49 received enalapril and 45 received placebo. Age, male/female ratio, duration of diabetes, and the other baseline data of the two groups are shown in Table 1. No statistically significant differences existed between the initial characteristics of the enalapril and the placebo groups.

During the first year of treatment, the urinary albumin excretion in the enalapril group decreased from an initial mean of 143 mg/24 h to an annual mean of 122 mg/24 h. Values greater than 300 mg/24 h were recorded in only two patients. Subsequently, a minimal but steady increase occurred in mean daily albumin excretion of these patients, with a fourth-year mean of 136 mg/24 h and a fifth-year mean of 140 mg/24 h. In six patients, albuminuria exceeded 300 mg/24 h. In contrast, among the placebo-treated patients, a steady, gradual increase occurred in mean daily albuminuria. The initial albumin value was 123 mg/24 h, the first-year mean was 134 mg/24 h, and the fifth-year value was 310 mg/24 h. Albumin values were greater than 300 mg/24 h in 19 patients and greater than 1 mg/24 h in three patients. The difference between the mean values of daily albuminuria in the two groups became statistically significant after the first year. However, the difference in the rate of change in proteinuria from baseline was statistically significant at the end of the first year (P < 0.05). These data are shown in Figure 1 and are numerically detailed in the Appendix Table. If the development of overt proteinuria ( 300 mg/24 h) is considered clinically important, the risk for developing this degree of proteinuria within 5 years of follow-up was 19/45 (42%) in the placebo group compared with 6/49 (12%) in the enalapril group. Therefore, enalapril treatment resulted in an absolute risk reduction of 30 percentage points for the development of overt proteinuria (95% CI, 15% to 45%; P < 0.001) for a 5-year period.

View larger version (18K): [in this window] [in a new window]   Figure 1. Proteinuria during 5-year follow-up in diabetics treated with enalapril or placebo. Each point on the curve represents the annual group average of 3 to 4 determinations per patient. Data are mean ±SD. Less proteinuria occurred in the enalapril group compared with placebo after the second year (P < 0.05 for the second year, P < 0.01 for the third year, and P < 0.005 for the fourth and fifth years).

Renal function, expressed as reciprocal creatinine (100/cr) remained unchanged in the enalapril group during the first 2 years of follow-up. From the third year onward, a small, nonsignificant but systematic decrease was recorded. The decrease was 1% of the initial value during the 5 years. In the placebo-treated group, a gradual, steady decrease of about 2% occurred in renal function each year. The difference between the initial and mean fifth-year values was 13% (P < 0.05). The difference between the mean rate of decrease of reciprocal creatinine among the enalapril- and the placebo-treated groups became significant after the second year. These data are shown in Figure 2 and are outlined in the Appendix Table.

View larger version (18K): [in this window] [in a new window]   Figure 2. Reciprocal creatinine (100/cr) levels expressed as percentage of initial value, during 5 years of follow-up in placebo-and enalapril-treated type II diabetics. Each value represents the annual average of the group composed of three to four determinations per year per patient. Values are mean ±SD. The mean rate of decline of reciprocal creatinine differed between the two groups (P < 0.05 for the second and third years, P < 0.02 for the fourth and fifth years).

The mean blood pressure in the enalapril-treated patients decreased slightly from an initial value of 99 mm Hg to 96 mm Hg during the first year. Thereafter, a gradual and nonsignificant increase was recorded; the final mean value was 100 mm Hg for the fifth year. In three patients in this group, systolic blood pressure values greater than 145 mm Hg or diastolic values greater than 95 mm Hg were recorded, and treatment with a long-acting nifedipine preparation (Pressolate), 10 to 20 mg twice daily, was added. In the placebo-treated group, the annual mean blood pressure increase was 1 to 3 mm Hg. In nine patients (2 each in the second to fourth years and 3 in the fifth year), elevated blood pressure levels were found and were treated by using long-acting nifedipine. The mean blood pressure values are shown in Figure 3.

View larger version (10K): [in this window] [in a new window]   Figure 3. Mean blood pressure values during 5-year follow-up in diabetic patients treated with enalapril or placebo. Each group mean represents 3 to 4 double determinations (blood pressure taken twice at same office visit) of mean blood pressure per year per patient. Values are mean ±SD. The group annual means did not differ (P = 0.14).

Changes compatible with diabetic retinopathy were found during the 5-year period in 9 patients (18%) who received enalapril and in 13 patients (29%) receiving placebo (P < 0.002). Two patients in each group had proliferative changes. The body mass index increased slightly in both groups of patients. The control of glucose levels was stable as judged by the steady, mean fasting blood sugar and glycosylated hemoglobin values during the 5 years. The serum potassium values were higher in the patients who received enalapril; the mean values during the fifth year were 4.26 ± 0.31 mmol/L for enalapril compared with 3.98 ± 0.36 mmol/L for placebo (P < 0.05).

Discussion

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This 5-year, prospective, double-blind study shows the long-term antiproteinuric effect of enalapril in normotensive patients with type II diabetes and microalbuminuria. Compared with placebo, the patients receiving enalapril also had effective protection of their kidney function. The difference between the enalapril- and the placebo-treated patients is important in view of the careful control of blood pressure in both groups using calcium-channel blockers when any increase of either systolic or diastolic pressure occurred beyond strictly defined normal limits.

Renal protection by angiotensin-converting enzyme inhibitors (albumin excretion and preservation of renal function) was described in hypertensive and in normotensive patients with diabetic nephropathy [7-15] and also in nondiabetic patients with proteinuria [24, 25]. However, most of the studies were short term, comprised small numbers of patients, and were mainly concerned with type I diabetes. Whether the beneficial effect of angiotensin-converting enzyme inhibitors would persist during a longer period was unknown.

In the present study, the decrease in proteinuria from an initial mean of 143 mg/24 h to a first-year average of 122 mg/24 h was followed by a slow increase toward a fifth-year average of 140 mg/24 h. Among the 49 patients treated with enalapril, only 6 patients had proteinuria that increased beyond the 300 mg/24 h threshold. In contrast, the mean daily albumin excretion of the patients who received placebo increased progressively from year to year. The fifth-year average was 152% greater than the initial value (P < 0.001), with 19 patients who had 24-hour albumin excretion values greater than 300 mg. These differences could not be attributed to differences in glycemic control, body mass index, or blood pressure values, which were very similar in both groups throughout the study period.

We monitored renal function using a crude method that is not as accurate as the method of measuring glomerular filtration rate used by others. Therefore, our conclusions about renal function must be expressed with appropriate caution. Nevertheless, for a group of patients who maintained a steady body mass index as well as an adequate and stable control of glucose metabolism, a steady trend in the plasma creatinine level is probably a reliable index of renal function [23, 26, 27].

Early stages of diabetes mellitus are characterized by increases in intracapillary pressure regardless of systemic arterial blood pressure [28]. Evidence, both in humans and in experimental animals, suggests that the effect of angiotensin-converting enzyme inhibitors in the kidney is to decrease efferent arteriolar resistance with resulting reduction of intraglomerular capillary hydraulic pressure [29-31]. In addition, a gradual decrease of glomerular membrane permeability to protein takes place. The dependence of these changes on the intraglomerular pressure is not clear. In certain animal models [32], the development of glomerulosclerosis was attenuated regardless of changes in intraglomerular pressure. Other studies [33] indicate that intraglomerular pressure is the main factor in preserving renal function.

Our present study corroborates previous evidence about the antiproteinuric effect and the renal protective effect of angiotensin-converting enzyme inhibition in early diabetic nephropathy in normotensive patients with type II diabetes. This effect is long-lasting and possibly additional to as well as independent of the antihypertensive effect of these agents. Thus, long-term administration of angiotensin-converting enzyme inhibitors should be considered seriously in both type I and type II diabetic patients with microalbuminuria, regardless of their blood pressure. We also need controlled, long-term clinical trials with these agents in diabetic patients without proteinuria to find a method for delaying the development of diabetic nephropathy.