Intrarenal Hemodynamic Changes After Captopril Test in Patients With Type 2 Diabetes: A duplex Doppler sonography study

A total of 40 normotensive Japanese patients with type 2 diabetes (aged 40–65 years) were enrolled. During the study period between May 1998 and April 1999, 232 diabetic patients without overt proteinuria were admitted to Osaka City University Hospital for treatment of diabetes or to attend a patient education course. Patients who met any of the exclusion criteria defined below were eliminated from the study, and the remaining patients who gave informed consent to participate in our study protocol were consecutively enrolled in the study. No patients were taking antihypertensive agents. The diagnosis of type 2 diabetes was established according to the Report of the Expert Committee on Diagnosis and Classification of Diabetes Mellitus (13). Patients met three additional criteria for inclusion: no episodes of ketoacidosis, no ketonuria, and insulin therapy (if any) initiated after ≥5 years of known disease. After admission to our diabetes ward, medical examinations were performed to exclude other renal diseases. Patients with nondiabetic or obstructive kidney disease and those with microscopic or macroscopic hematuria, abnormal urinary sediment, history of glomerulonephritis or nephroureterolithiasis, dilated renal pelvis or atrophied kidney on ultrasonography, overt proteinuria, or elevated serum creatinine concentration (>1.2 mg/dl) were excluded. No patients had a clinical history or signs of cerebrovascular disease, peripheral vascular disease, or cardiovascular disease. During admission, each patient received a special diet (27–30 kcal/kg ideal body wt/day) that consisted of 50% carbohydrate, 30% fat, 20% protein, and 10 g salt per day. The study design was approved by the hospital committee on ethics. Each subject gave informed consent before entering the study. As a control, 15 age- and sex-matched subjects who visited our hospital for medical screening and gave informed consent were consecutively recruited from outpatient clinics of our hospital during the study periods. Inclusion criteria for nondiabetic control subjects were as follows: systolic blood pressure <130 mmHg and diastolic blood pressure <85 mmHg; fasting plasma glucose <126 mg/dl; no clinical history of myocardial infarction, cerebral infarction, or peripheral vascular disease; and no use of medication likely to affect renal or systemic hemodynamics.

For each diabetic patient, 24-h urine samples were collected on three consecutive days to determine the level of urinary albumin excretion (UAE) and creatinine clearance. In each patient, the level of 24-h UAE was the mean value for the 3 consecutive days. To examine the difference in responsiveness to captopril between the patients with normoalbuminuria and those with microalbuminuria, the patients were predefined as being in one of two groups: group 1, consisting of patients with UAE <30 mg/day (n = 20); and group 2, consisting of patients with UAE ≤30 or <300 mg/day (n = 20).

Blood pressure was recorded three times after a subject had rested in the supine position for at least 15 min. A standard mercury sphygmomanometer with a cuff that adapted to arm circumference was used. The systolic blood pressure was considered the point of first audibility of Korotkoff sounds, and the diastolic blood pressure was considered the point at which the Korotkoff sounds disappeared. The three measurements were averaged. In each patient, blood pressure was measured both before and 1 h after oral administration of 25 mg captopril.

Information on smoking habits was obtained by a self-administered questionnaire. Lifelong exposure to smoking was estimated as the product of years of smoking and the number of cigarettes smoked daily at the time of the study (cigarette-years).

Blood samples were collected after an overnight fast for analysis of serum concentrations of creatinine, total cholesterol, triglycerides, and HDL cholesterol by standard laboratory methods. Plasma levels of glucose were measured by the glucose oxidase method, and HbA_1c was measured by high-performance liquid chromatography (HI-AUTO A1C; Sekisui Chemical, Osaka, Japan). The level of urinary albumin was measured in 24-h urine collections by immunoturbidmetry (TIA MicroAlb Kit; Nittobo, Tokyo, Japan). The UAE rate was expressed in milligrams per 24 h.

Blood samples for measuring plasma renin activity (PRA) and plasma aldosterone (PA) were collected after subjects had remained in the supine position for at least 15 min. PRA and PA were measured by radioimmunoassay (Dainabot, Tokyo, Japan). PRA and PA were measured both before and 1 h after oral administration of 25 mg captopril.

To measure the RI, patients and healthy control subjects were asked to lie down for at least 15 min before the examination. RI was measured as previously reported (10,11,14). In brief, the peak systolic flow velocity (PSV) and the end-diastolic flow velocity (EDV) were automatically calculated using the ultrasound apparatus. Flow velocities were determined from signals that were stable for at least five pulse beats, and measurements represented the average of five complete waveforms. The resistance parameter, RI, was determined as follows (10,11,14): RI = (PSV - EDV)/PSV.

Three different interlobar arteries from the right kidney were randomly selected and examined, and the mean value was calculated. The coefficient of variance for RI was 3.6%, as we reported previously (10,11).

The same procedure was followed 1 h after subjects were given 25 mg captopril orally. To avoid possible side effects, subjects were kept in the supine position and blood pressure was monitored every 30 min until the end of the study.

To evaluate the changes in RI after the captopril test, ΔRI was calculated by the following formula: (RI at 1 h after captopril administration) - (RI at baseline).

Values are expressed as mean ± SD. Values for clinical parameters were compared among three groups by one-way ANOVA with Scheffe’s F test. Univariate χ² analysis was used for comparison by sex. The difference between values before and after captopril administration was examined by paired Student’s t test. The relationships between ΔRI and biochemical variables were examined by linear regression analyses. A stepwise multiple regression analysis with forward elimination method was performed to evaluate the factors affecting ΔRI in patients with type 2 diabetes. The following independent variables were included in the model: age, BMI, duration of diabetes, smoking (cigarette-years), HbA_1c, total and HDL cholesterol, triglycerides, baseline mean blood pressure, and baseline PRA and PA as continuous variables; sex (female = 0, male = 1) was included as a categorical variable. The F value was set at 4.0 at each step. All statistical analyses were performed using Stat-View V software (Abacus Concepts, Berkley, CA) for a Macintosh computer. A level of P < 0.05 was accepted as statistically significant.

The clinical characteristics of diabetic patients and control subjects are shown in Table 1. Diabetic patients were grouped by level of UAE. Among the three groups, no significant differences were found in sex, age, BMI, cigarette-years, systolic or diastolic blood pressure, creatinine, total cholesterol, triglycerides, or HDL cholesterol. Fasting plasma glucose was significantly higher in diabetic patients than in control subjects. There were no significant differences in the duration of diabetes, fasting plasma glucose, or HbA_1c between the two groups of diabetic patients.

The changes in blood pressure, heart rate, PRA, and PA from before to after the captopril test are shown in Table 2. Systolic and diastolic blood pressures after the captopril test were significantly lower than before the test in both the control subjects and the two groups of diabetic patients (P < 0.001). In the control subjects, PRA after the captopril test was significantly higher than before the test (P < 0.01). In diabetic patients, PRA in group 1 was significantly higher after the captopril test than before it (P < 0.01), and in group 2 PRA was higher with borderline significance (P = 0.06). PA after the captopril test was significantly lower than that before the test in all three groups (P < 0.01 in control subjects; P < 0.05 in diabetic patients).

No significant difference existed in baseline RI values among the three groups (Table 2), although RI values in diabetic patients tended to be higher than those in control subjects, as previously reported (10). In control subjects, the RI values after the test were significantly higher than those before the test (P < 0.01). RI values after the captopril test were significantly lower than those before the test in both groups with diabetes (P < 0.001), in contrast to the control subjects. There were no significant differences in RI values between the two groups of diabetic patients, either before or after the test (unpaired Student’s t test).

Changes in RI value (ΔRI) are shown in Fig. 1. There were significant differences in ΔRI between the control subjects and group 1 of the diabetic patients (P < 0.0001) and between the control subjects and group 2 of the diabetic patients (P < 0.0001, unpaired Student’s t test). There was no significant differences in ΔRI between the two groups of diabetic patients (unpaired Student’s t test).

Linear regression analyses were performed to examine the relationships between ΔRI and clinical parameters. Between ΔRI and HbA_1c in diabetic patients, there was a negative correlation with a coefficient of r = −0.458 (P < 0.005, Fig. 2). Between ΔRI and baseline PRA in patients, there was a negative correlation with a coefficient of r = −0.339 (P < 0.05). However, there was no significant correlation between ΔRI value and other clinical parameters in diabetic patients, including age, duration of diabetes, BMI , cigarette-years, blood pressure, total cholesterol, triglycerides, HDL cholesterol, and baseline PA. There was no significant correlation between ΔRI and changes in mean blood pressure, PRA, or PA in diabetic patients either.

Results of a multiple regression analysis examining possible predictors independently affecting ΔRI in diabetic patients are shown in Table 3. HbA_1c and baseline PRA value significantly and independently affected ΔRI in diabetic patients (R² = 0.391, P < 0.0001)

In the present study, we examined intrarenal hemodynamic changes after the captopril test using duplex Doppler sonography to investigate the response to ACE inhibitors in patients with type 2 diabetes. We found a significant decrease in RI after the captopril test in patients with type 2 diabetes, in contrast to control subjects, in whom RI values were significantly increased. Poor control of blood glucose, as represented by increased HbA_1c, and basal PRA affected the magnitude of decrease in RI in patients with type 2 diabetes.

Veglio et al. (15) reported that in healthy subjects and patients with mild hypertension, RI values of interlobar arteries after the captopril (50 mg) test were significantly higher than those before the captopril test. In the present study, RI values in the control subjects were significantly increased after the captopril test, being consistent with the results of Veglio et al., despite the difference between studies in the dose of captopril used. A dose of 25 mg captopril was chosen for the present study, because of differences in body size between Caucasians and Japanese. Although the precise mechanism of increase in RI value is unknown, the increase in RI values in the control subjects could be related to functional vasoconstriction in the kidney (autoregulation), which may be induced by significant decrease in systemic blood pressure induced by captopril (15).

In contrast to the increase in RI values after the captopril test in control subjects, a significant decrease in RI after the captopril test was seen in diabetic patients. Our results suggest the disruption of autoregulation in the kidney against decrease in blood pressure is present in diabetic patients. Although disruption of renal autoregulation was also reported in patients with advanced hypertension (15), our patients were all normotensive, suggesting that other mechanisms of decrease in RI values are present in diabetic patients. Because the average salt consumption by Japanese adults was 12.2–13.2 g/day during the 1990s, differences in salt intake between control subjects and diabetic patients (10 g/day) probably did not account for differences in RAS activity between control subjects and diabetic patients.

Examining 22 patients with renovascular hypertension, Veglio et al. (12) demonstrated that RI values after the captopril test were significantly decreased in kidneys with stenotic arteries but not in kidneys with nonstenotic arteries. This phenomenon is believed to be due to the dependency of the intrarenal vasculature on increased RAS activity in ischemic kidneys. In the present study, we found that one of the significant factors affecting the magnitude of decrease in RI value was baseline PRA. The higher the basal PRA was, the greater decrease in RI value after the captopril test was seen. These results suggest that diabetic kidneys may also depend on RAS activity, as seen in the ischemic kidneys of patients with renovascular hypertension. They also suggest that RAS activation may be present in the kidney of diabetic patients.

In fact, both experimental and clinical studies have shown that hyperglycemia induces RAS activation, leading to increase in renal vascular resistance. Woods et al. (16) demonstrated that intrarenal infusion of glucose in anesthetized dogs increased renin secretion. Using hyperglycemic clamp, Miller et al. (17) showed that, in patients with type 1 diabetes, hyperglycemia increased PRA and renal vascular resistance. Furthermore, recently, the angiotensin II type 1 receptor antagonist losartan was demonstrated to significantly increase renal plasma flow and significantly decrease renal vascular resistance in patients with type 1 diabetes, suggesting increased activity of the intrarenal RAS in diabetic patients (3). Price et al. (4) also found increased intrarenal RAS activity in type 2 diabetic patients with overt proteinuria, compared with healthy subjects, by demonstrating that a significant increase in renal plasma flow was induced by another angiotensin II type 1 receptor antagonist irbesartan. Mizuiri et al. (18) demonstrated increased immunostaining of angiotensin converting enzyme in the diabetic glomeruli, suggesting increased RAS activity in diabetic kidney. These reports, together with the present study, suggest that intrarenal hemodynamics change and become dependent on RAS activity in the presence of increased intrarenal RAS activity in diabetic patients. It has been reported that activation of RAS by hyperglycemia increases the renal vascular resistance in both type 1 and type 2 diabetes (3,17). In the present study, patients with relatively higher HbA_1c were included because they were treated for poor glycemic control, possibly leading to increased RAS activation. The decrease in RI values observed after the captopril test in the present study could be caused by elimination of intrarenal vasoconstriction after ACE inhibition. In the present study, the response to captopril (decrease in RI) was significantly affected by HbA_1c level. This result further indicates that intrarenal RAS activity is increased as glycemic control becomes poorer, consistent with the findings of the previous study (3,17). In the present study, there were no significant differences in response to captopril between patients with normoalbuminuria (group 1) and those with microalbuminuria (group 2).

The results of several studies, along with those of the present study, suggest that activation of the intrarenal RAS may be present in diabetic patients. The present study demonstrated that activation of intrarenal RAS could be caused by poor glycemic control and that blockade of RAS activation by ACE inhibition significantly reduced renal vascular resistance. By examining intrarenal hemodynamic changes, the present study has emphasized that relief from intrarenal RAS activation by strict control of blood glucose and/or use of RAS inhibition is important in improvement of renal hemodynamics and has provided evidence of a beneficial effect of ACE inhibition in diabetic patients.