Introduction

Hypertension is a common comorbidity of type 2 diabetes mellitus (T2DM), and both are associated with an increased cardiovascular risk [1, 2]. Current guidelines emphasize strict hypertension treatment in diabetic patients [3], and tight blood pressure (BP) management has even been suggested to provide greater benefit than strict glucose control [4].

Nocturnal BP usually drops by 10% or more of daytime BP [5]. However, despite well-controlled hypertension and strictly regulated diabetes, nondipping status, defined as a nocturnal decline in BP of less than 10% as an expression of an abnormal circadian BP rhythm [5], remains an enduring cardiovascular risk factor in T2DM patients [6, 7]. Cardiovascular autonomic neuropathy (CAN) and endothelial dysfunction are the principal mechanisms put forward in explanation of the blunted nocturnal BP decline in T2DM patients [8,9,10]. In diabetic patients with CAN, the normal circadian sympathovagal balance has become affected, with blunted parasympathetic heart rate (HR) control coinciding with enhanced sympathetic vasomotor activation during the night [11].

In subjects with essential hypertension, nondipping status has been alleged to be associated with endothelial dysfunction [12], whereas a similar relationship was established in patients with therapy-resistant hypertension [13]. The observation that such a relationship was also found in patients with type 1 diabetes [9] suggests that vascular impairment contributes to the impaired nocturnal BP response. In T2DM, the incidence of CAN vs. microvascular complications is lower, whereas the prevalence of the nondipping pattern amounts to ~30% [14]. We hypothesized that, in T2DM subjects, the degree of microvascular disease rather than CAN interferes with the normal nocturnal reduction in BP. We further postulated that in T2DM patients without overt signs of either microvascular disease or CAN, a normal nocturnal dipping pattern is present. To address these aims, subjects with T2DM and microvascular damage (DM+) underwent 24-hour ambulatory BP measurements, preceded by cardiovascular autonomic function testing and measurement of baroreflex sensitivity. T2DM subjects without microvascular complications (DM−) and control subjects without T2DM (CTRL) served as reference groups.

Methods

Subjects and study design

Twenty-two T2DM subjects with microvascular complications (DM+, aged 57 ± 8 years), 23 subjects without microvascular complications (DM−, 54 ± 8 years), and 16 reference subjects without T2DM matched for age, sex, and ethnicity (CTRL, 54 ± 8 years) were consecutively recruited from the outpatient clinic (Table 1). Verbal and written information was given to each subject concerning the study objectives and measurement techniques. Informed consent was obtained from each subject as approved by the Academic Medical Center Medical Ethical Committee, and the experiments were performed in accordance with the Declaration of Helsinki.

Table 1 Baseline characteristics

Subjects were diagnosed with T2DM according to the WHO criteria and were treated with insulin and/or oral antihyperglycemic agents. Selection criteria for the DM+ group included microvascular complications such as proliferative or nonproliferative retinopathy, microalbuminuria, or polyneuropathy. Retinopathy was confirmed by an ophthalmologist. Microalbuminuria was defined as a persistent urinary albumin excretion rate of 30-300 mg/24 h or albumin/creatinine ratio > 2.5 mg/mmol for men or > 3.5 mg/mmol for women in the absence of clinical or laboratory evidence of other kidney or renal tract disease. Subjects without these complications were included in the DM− group. Exclusion criteria included presence of CAN, history of stroke, uncontrolled or resistant hypertension, defined as BP remaining above goal despite concurrent use of 3 antihypertensive agents of different classes [15], or chronic kidney disease stage 4 or 5 with or without macroalbuminuria.

Study protocol and instrumentation

The subjects were requested to abstain from caffeinated beverages 12 h prior to reporting in the morning to the laboratory (room temperature 22 °C). After instrumentation and 20 min of supine rest, baseline systemic variables were recorded. Continuous BP was measured noninvasively by finger photoplethysmography (Portapres, Finapres Medical Systems, Amsterdam, The Netherlands) with the cuff placed on the middle phalanx of the left middle finger kept at heart level and was calibrated by an automated noninvasive BP-measuring device (HEM-7000-E, Omron, Kyoto, Japan). HR was monitored using a 3-lead ECG. The Valsalva strain was measured by a pressure transducer (Hewlett-Packard 1290, Waltham, MA, U.S.A.) connected to a mouthpiece. A marker pulse identified the onset of the different maneuvers. For offline analysis, all signals were analog-to-digital converted at a sampling rate of 200 Hz and stored on disk. Prior to 24-hour ambulatory BP measurements, cardiovascular autonomic function testing and baroreceptor sensitivity measurements were performed in all patients.

Cardiovascular autonomic function

Parasympathetic control of HR was evaluated by forced respiratory sinus arrhythmia (FRSA) at a 0.1-Hz breathing frequency and by quantifying the time-course and magnitude of HR responses expressed as the ratio of the maximal and minimal HR in the first 30 s (HRmax/min) to active standing and the Valsalva maneuver [16]. Efferent sympathetic vasomotor function was assessed by the BP responses to active standing and the Valsalva maneuver. A mouthpiece was connected to a manometer, and the subjects were instructed to maintain an expiratory pressure of 40 mm Hg for 10 s. A small-bore needle was inserted into a rubber stopper to prevent the subjects from maintaining manometer pressure by closing the glottis. Care was taken to prevent deep breathing before and after the release of strain [17]. The standing up maneuver was performed within 2–3 s during a normal inspiration after a verbal command to start was given at the end of expiration [18].

Orthostatic hypotension was defined as a fall in systolic BP of at least 20 mm Hg or diastolic BP of at least 10 mm Hg. The presence of two or more abnormal test results was considered to reflect the presence of CAN [19].

Baroreceptor sensitivity

Afferent, central, and vagal efferent baroreceptor reflex pathways were evaluated by quantifying time-domain cardiac baroreflex sensitivity (xBRS) using the cross-correlation time-domain method [20]. Beat-to-beat values of systolic BP and interbeat interval were interpolated and resampled at 1 Hz. Correlations between systolic BP and interbeat interval were computed using a sliding 10-second window, with delays of 0–5 s for interval [20]. The highest coefficient of correlation was selected and accepted if P < 0.01. xBRS was the slope of the regression line between changes in interbeat interval vs. systolic BP, expressed as ms.mm Hg−1. To evaluate baroreflex adaptation to a physiological perturbation, such as postural stress, xBRS was measured in both the supine and upright positions.

24-hour ambulatory blood pressure

Thereafter, all subjects underwent 24-hour oscillometric ambulatory BP measurements (Mobilograph, APC, Belfeld, The Netherlands). BP measurements were recorded at 15-min intervals during the daytime and at 30-min intervals during the night. The 24-hour ambulatory BP measurement device was calibrated at the start of the study by a noninvasive BP-measuring device (HEM-7000-E, Omron, Kyoto, Japan). Subjects were asked to record their daily activities in diaries, including medication intake, meals, exercise, and sleeping period. Nocturnal period was determined according to patients’ diaries. Average BP was calculated for both daytime and night. The nocturnal BP change was calculated as percent changes in both systolic and diastolic BP relative to daytime systolic and diastolic BP set as baseline. Nondipping status was defined as a nocturnal decrease of less than 10% in systolic or diastolic BP [21]. Short-term BP and HR variability are expressed as the standard deviation (SD) of their average at daytime and nighttime. In addition, BP variability is expressed as the coefficient of variability, calculated as SD/mean pressure x 100%. Pulse pressure is expressed as the difference between systolic and diastolic BP.

Data analysis

Data are presented as the means (±SD) and differences between groups were identified by unpaired Student's t-test when data fitted a normal distribution and by a Mann–Whitney rank-sum test when data were not normally distributed. Differences among the three groups were identified by ANOVA. P < 0.05 was considered to indicate a statistically significant difference. Data analyses were performed using SigmaStat software (version 3.1, Systat Software Inc., San Jose, CA, USA) and Stata (version 14.2, StataCorp LLC, College Station, TX, USA).

Results

Group characteristics

The group characteristics are listed in Table 1. The groups were comparable for gender ratio, age, BMI, and serum creatinine level. Known duration of diabetes was not different in the DM+ vs. DM− groups (11.6 ± 6.8 vs. 9.2 ± 4.1 years, NS). DM+ subjects had higher HbA1c (P < 0.05 vs. DM−), with higher levels of albuminuria (P < 0.05 vs. CTRL and P < 0.01 vs. DM−) and albumin/creatinine ratio (P < 0.05 vs. DM−). Among the DM+ patients, 10 had microalbuminuria, with diabetic retinopathy in 11 subjects (7 with nonproliferative diabetic retinopathy), whereas 16 patients suffered from polyneuropathy. Although the standing HRmax/min was lower in the DM+ group (1.17 ± 0.10 vs. 1.26 ± 0.14 in DM− and 1.34 ± 0.23 in CTRL, P < 0.05), no differences were found in Valsalva HRmax/min and FRSA, indicating intact parasympathetic HR control, whereas sympathetic vasomotor function was not impaired in any of the subjects.

24-hour ambulatory BP monitoring

The average 24-hour systolic and diastolic BP and baseline HR at daytime were comparable between groups (Table 2). The nocturnal BP reduction as observed in the CTRL group was smaller in the DM− group and was further blunted in the DM+ group, with more nondippers in the latter group (64% vs. 39% in DM− and 19% in CTRL). No differences in supine nocturnal period were observed among the groups. The nocturnal reduction in systolic and diastolic BP was lower in DM+ subjects (8 ± 5%/11 ± 6% vs. 12 ± 6%/14 ± 5% in DM−, P < 0.05, and vs. 17 ± 5/19 ± 7% in CTRL, P < 0.01, Fig. 1). In addition, the nocturnal BP dip was reduced in DM− compared with CTRL (P < 0.05). More subjects in the DM+ group were treated with ACE inhibitors, diuretics, and calcium channel-blocking agents compared with the CTRL and DM− groups. Additionally, more subjects in both DM groups received statin therapy.

Table 2 Characteristics of 24-hour ambulatory BP and medication
Fig. 1
figure 1

Group-averaged nocturnal dips in systolic (left panel) and diastolic (right panel) blood pressure in DM+ (black bars), DM− (gray bars), and CTRL (white bars) subjects. Values are means ± SD for n = 16 (CTRL), n = 23 (DM−), and n = 22 (DM+)

BP variability and cardiac baroreflex sensitivity

Variabilities of systolic and diastolic BP and HR were comparable among groups throughout the 24-hour period (Fig. 2 and Table 2). The SD of systolic BP was smaller at night vs. daytime for all groups, whereas the SDs of both diastolic BP and HR were reduced during the nighttime only for the CTRL subjects (P < 0.05). Nighttime pulse pressure declined in both the CTRL and DM− subjects but not in the DM+ group, indicating persistently higher pulse pressure values throughout the day. xBRS was reduced in the DM− group in the supine position (7.7 ± 3.3 vs. 12.3 ± 8.3 ms.mm Hg−1 in CTRL, P < 0.05) and was further impaired in the DM+ group (4.6 ± 2.0 ms.mm Hg−1, P < 0.01 vs. CTRL and DM−; Fig. 3).

Fig. 2
figure 2

Group-averaged standard deviation (SD) in systolic (left upper panel) and diastolic (right upper panel) blood pressure, heart rate (left lower panel), and pulse pressure (right lower panel) in DM+ (black bars), DM− (gray bars), and CTRL (white bars) subjects. **P < 0.01 vs. CTRL, †P < 0.05 vs. DM−. Values are means ± SD for n = 16 (CTRL), n = 23 (DM−), and n = 22 (DM+)

Fig. 3
figure 3

Group-averaged baroreflex sensitivity (xBRS) in the supine and upright positions in DM+ (black bars), DM− (gray bars), and CTRL (white bars) subjects. *P < 0.05 and **P < 0.01 vs. CTRL, †P < 0.05 and ‡P < 0.01 vs. DM−. Values are means ± SD

Discussion

The findings of this study provide novel insight into the diurnal BP behavior in patients with T2DM. In T2DM patients with clinically manifest microvascular disease (DM+), blunting of the nocturnal BP dip coincided with a persistently enlarged arterial pulse pressure in the absence of CAN. In addition, the nocturnal BP decline was smaller in the DM patients without (DM−) microvascular complications compared with the CTRL group. Furthermore, baroreflex sensitivity was reduced to a larger extent in the DM+ subjects compared with DM− subjects. Taken together, these findings indicate that the nocturnal BP dipping status is associated with progression of microvascular disease rather than CAN.

An impaired nocturnal decline in BP in T2DM patients has been linked to a variety of factors, including CAN, diabetic nephropathy, and poor glycemic control [8, 22]. Nondipping BP status is considered to be a manifestation of an abnormal circadian BP rhythm [5] related to a shift in the sympathovagal balance towards enhanced sympathetic activation but attenuated vagal activity [11]. Impairment of parasympathetic HR control is an early manifestation of CAN in DM patients [16, 23]. In the DM+ group, reduced baroreflex sensitivity per se may reflect reduced parasympathetic cardiovascular function. However, resting HR did not differ between groups, and, together with the normal HR responses during forced breathing, integrity of parasympathetic HR control was confirmed. Normal BP responses to the Valsalva maneuver and to active standing in DM+ also signify integrity of sympathetic vasomotor function. Absence of manifest CAN in this study disputes the proposed role of CAN [24] as the mechanism of blunted nocturnal BP decline in both type 1 and 2 diabetes mellitus. The duration and magnitude of hyperglycemia affect both microvasculature and neuronal integrity. Although prevalence of CAN increases with diabetes duration, in the Rochester diabetic study, CAN was detected in no more than approximately 7%, whereas half of the patients presented with distal symmetric sensorimotor polyneuropathy [25], which is considered the most common type of diabetic neuropathy. Although in our study, none of the subjects fulfilled the criteria of CAN, defined as the abnormality of at least two cardiovascular autonomic test results, the observed discrepancy with the reduction of baroreflex sensitivity in both DM groups remains unexplained.

The finding that an increased nocturnal systolic BP precedes the development of albuminuria [26] has been attributed to consequent hemodynamic alterations, including increased renal blood flow and glomerular hyperfiltration [8]. This suggests that the nondipping status, i.e. nocturnal hypertension, is the cause rather than a result of microalbuminuria, which is also considered a marker of a chronic inflammatory state and cardiovascular disease risk [27]. The present finding that in some T2DM subjects with diabetic retinopathy or polyneuropathy without microalbuminuria, the nocturnal BP decline was blunted despite intact autonomic cardiovascular function, rather points to microvascular disease itself being involved in the development of nocturnal hypertension. The nondipping BP status is recognized as an independent risk factor for cardiovascular disease [5,6,7], but whether the nondipping pattern should be considered as the cause rather than the effect of target organ damage remains to be determined [28].

Nocturnal hypertension is a common phenomenon in patients with chronic kidney disease and salt-sensitive hypertension. It is associated with diminished renal sodium excretory capability that manifests when salt intake is high [29]. In these patients, the high nocturnal BP is considered to compensate for the reduced capacity to excrete sodium during the daytime by enhancing pressure natriuresis during the night [30]. Although renal function was comparable among groups and patients with overt proteinuria were excluded, we did not control for dietary sodium intake. The percentage of patients taking antihypertensive medication was larger in the DM groups, but after adjustment, the differences in the magnitude of nocturnal BP dipping among the DM vs. control subjects persisted.

The present study suggests blunting of the nocturnal BP dip as an early manifestation of microvascular disease. In contrast to our expectations, no difference in BP variability was observed among groups throughout the 24-hour period, notwithstanding the progressive decline in baroreflex sensitivity in DM subjects. Together with an absent relationship between nocturnal BP decline and baroreflex sensitivity, the contribution of baroreflex sensitivity to short-term BP variability seems limited.

Limitations

There are potential limitations that need to be considered. The dipping status is influenced by the quality of sleep [31]. All subjects recorded their daily activities, including sleeping period, although we did not measure the quality of their sleep. We are aware that the nondipping pattern is also present in patients with good sleep quality according to their diary input [32], but we cannot exclude a potential influence of differences in quality of sleep among groups, if any, to the findings of our study.

Sustained hyperglycemia has both cerebral and systemic hemodynamic effects. In healthy subjects, increases in cerebral blood flow velocity and limb blood flow were demonstrated during sustained hyperglycemia without alterations in BP and sympathetic tone [33, 34]. In addition, in newly diagnosed T2DM patients, acute hyperglycemia results in an increase in BP that is reversed by l-arginine infusion. This suggests a reduced nitric oxide availability as one of the underlying mechanisms [35]. In the DM+ group, a larger HbA1c level coincided with the presence of microvascular complications. Traditionally, microvascular disease is considered a long-term complication of sustained hyperglycemia. The contribution of the higher HbA1c as a reflection of poor glycemic control in itself is unclear. In patients with obesity-related prediabetes, HbA1c and C-reactive protein were shown to be independently associated with blunted nocturnal BP dipping [36]. Pistrosch et al. demonstrated that the nondipping status was associated with higher postprandial plasma glucose levels, with the background of similar HbA1c and fasting plasma glucose in the dippers [37]. We are not aware of any glucose clamp studies evaluating the direct effects of sustained hyperglycemia on the nocturnal BP pattern. In spite of the problem of relating the abnormal BP pattern to sustained hyperglycemia vs. microvascular disease, our finding of impaired nocturnal BP dipping in DM patients in absence of CAN remains unchallenged.

Perspectives

The nondipping status increases the risk of developing cardiovascular disease independently of office BP [38]. In addition, an increased pulse pressure is associated with a greater cardiovascular risk than systolic BP [39]. Widening in pulse pressure with aging has been attributed to arterial stiffness, mainly leading to an increase in systolic BP [40]. Arterial stiffness may have contributed to the altered circadian BP rhythm with increased pulse pressure observed in the DM patients. The clinical implication is that, despite strict BP management and glycemic control in DM patients, nocturnal hypertension with persistently increased pulse pressure throughout the night may be a residual risk factor contributing to the increased cardiovascular risk profile. The MAPEC, HOPE, and Syst-Eur studies have demonstrated that bedtime administration of antihypertensive agents converted the 24-hour BP pattern to a dipping pattern, reducing the progression of target organ damage and reducing the cardiovascular risk [41,42,43], which was also demonstrated in T2DM patients [44]. Furthermore, SGLT2 inhibitors in T2DM patients were shown to reduce the incidence of cardiovascular events and were apparently associated with improvement in the BP dipping pattern [45]. We consider that additional information regarding the dipping status and pulse pressure is of great importance for cardiovascular risk assessment; therefore, we recommend the use of 24-hour ambulatory BP monitoring in clinical practice in addition to office BP measurements.