Reduced Systemic Microvascular Function in Patients With Resistant Hypertension and Microalbuminuria: an Observational Study

doi:10.21203/rs.3.rs-4159503/v1

Resistant hypertension (RH) may be associated with microalbuminuria (MAU), a marker of increased mortality, and both may be related to microvascular damage. Laser speckle contrast imaging (LSCI) is an innovative approach for noninvasively evaluating systemic microvascular endothelial function useful in the context of RH with or without MAU. Microalbuminuria was defined as a urine albumin-to-creatinine ratio between 30 and 300 mg/g. Microvascular reactivity was evaluated using LSCI to perform noninvasive measurements of cutaneous microvascular perfusion changes. Pharmacological (acetylcholine [ACh], or sodium nitroprusside [SNP]) and physiological (postocclusive reactive hyperemia [PORH]) stimuli were used to evaluate vasodilatory responses. Thirty-two patients with RH and a normal urine albumin-to-creatinine ratio (RH group) and 32 patients with RH and microalbuminuria (RH + MAU) were evaluated. Compared with patients without MAU, patients with RH + MAU showed reduced endothelial-dependent systemic microvascular reactivity, as demonstrated by an attenuation of microvascular vasodilation induced by PORH. On the other hand, ACh-induced vasodilation did not differ between groups. The results also revealed reduced endothelial-independent (SNP-induced) microvascular reactivity in hypertensive patients with MAU compared with patients without MAU. In this study, there was evidence of endothelial dysfunction associated with impaired microvascular smooth muscle function in patients with RH + MAU. This may suggest that patients with RH need more intensive therapeutic strategies for the control of blood pressure to avoid further vascular damage and the resulting consequences.

Health sciences/Diseases/Cardiovascular diseases

Health sciences/Medical research

microvascular dysfunction

resistant hypertension

laser speckle contrast imaging

In some cases, systemic arterial hypertension can progress to a phenotype called resistant arterial hypertension.
Nondiabetic microalbuminuria is a biomarker of endothelial dysfunction that contributes to both atherosclerotic macrovascular disease and renal microvascular disease.

The present study provides evidence of microvascular dysfunction in patients with resistant arterial hypertension and microalbuminuria, with altered endothelium-dependent and endothelium-independent vasodilatation, compared to patients without microalbuminuria.
These findings may suggest that additional risk stratification strategies for identifying microvascular dysfunction might be useful in patients with resistant arterial hypertension and that more intensive therapeutic strategies for the control of blood pressure and associated conditions.

Systemic arterial hypertension (SAH) is the result of complex interactions between environmental, genetic and social factors that initiate and perpetuate blood pressure elevations and is one of the most prevalent cardiovascular diseases worldwide (1, 2). SAH is characterized by important changes in systemic microvascular reactivity and density, as demonstrated by studies reporting altered microvascular endothelial responses to a variety of stimuli in hypertensive patients, identified mostly via laser microvascular perfusion monitoring technology (3-6). Given that microvascular dysfunction and rarefaction are unquestionably involved in the pathophysiology of SAH and that hypertension can lead to microvascular alterations, a bidirectional causality most likely exists between the two conditions (7).

In some cases, SAH can progress to a phenotype called resistant arterial hypertension (RH) (8). RH is defined as either the inability to achieve blood pressure control despite the administration of at least three antihypertensive medications at maximal or maximally tolerated dosages, including a diuretic, or controlled blood pressure requiring at least four medications (9). Typical three-drug regimens include a renin-angiotensin system inhibitor, a calcium channel blocker, and a thiazide or thiazide-like diuretic (9).

Microalbuminuria (MAU) is defined as an abnormally increased excretion rate of albumin in the urine in the range of 30-299 mg/g creatinine and is associated with increased mortality independent of the estimated glomerular filtration rate (eGFR) (10, 11). MAU is associated with different cardiometabolic conditions, including salt sensitivity and hypertension, obesity, insulin resistance and diabetes, as well as other components of metabolic syndrome, such as dyslipidemia (12). Moreover, MAU indicates microvascular injury and is independently associated with a greater risk of major adverse cardiovascular events and all-cause mortality in hypertensive patients, especially in those with RH (13-15).

As a marker of microvessel injury, MAU may be associated with other microvascular damage indicators, such as endothelial dysfunction, as demonstrated by different methods. Laser speckle contrast imaging (LSCI) provides an innovative approach for noninvasively evaluating systemic microvascular endothelial function, with physiological (postocclusive reactive hyperemia) or pharmacological (acetylcholine and sodium nitroprusside) stimulation to evaluate systemic endothelial-dependent and endothelial-independent microvascular reactivity (16). A major advantage of this technique is that LSCI is more reproducible than earlier procedures, such as laser Doppler flowmetry and laser Doppler imaging (17). LSCI has also previously been shown to be an effective noninvasive technique for evaluating systemic microvascular reactivity in patients who present with cardiovascular and metabolic diseases (16, 18).

Therefore, this study aimed to evaluate systemic microvascular function in patients with RH, with or without MAU, to identify possible associations between MAU and endothelial dysfunction in the context of RH. The combination of the two alterations (MAU and endothelial dysfunction) might indicate a greater risk profile, which could merit more intensive therapeutic interventions to improve patient prognosis.

Study design

The present study was performed in accordance with the Helsinki Declaration of 1975, revised in 2000, and was approved by the Institutional Review Board (IRB) of the National Institute of Cardiology, Rio de Janeiro, Brazil, under protocol # CAAE 53946021.8.0000.5272. Once deemed eligible to participate in this study, all subjects read and signed an informed consent document approved by the IRB. The study was registered at ClinicalTrials.gov (https://register.clinicaltrials.gov) under protocol # NCT05464849, initial release 12/07/2022.

Sixty-four resistant hypertensive outpatients of both sexes who were eligible for this study were consecutively recruited from our hypertension clinics and underwent complete medical examinations. All patients had a previous diagnosis of RH and were monitored by the institution's specialized outpatient clinic. Before inclusion, secondary causes of hypertension were ruled out, and blood pressure levels were documented through 24-hour ambulatory blood pressure monitoring.

Microalbuminuria was defined as a urine albumin-to-creatinine ratio (UACR) between 30 and 300 mg/g in a routine spot urine sample (19, 20). In the present study, there were 32 resistant hypertensive patients with a normal UACR (RH group) and 32 resistant hypertensive patients who presented with microalbuminuria (RH+MAU). The estimated glomerular filtration rate was calculated using the chronic kidney disease epidemiology collaboration (CKD-EPI) creatinine equation (21).

All evaluations were performed in the morning between 8 AM and 12 PM after a 12-hour fast. Blood specimens were collected, and the subjects were asked to lie down for 20 minutes in a quiet environment that had a constant temperature of 23 ± 1 °C.

Evaluation of skin microvascular flow and reactivity

The microcirculatory tests were performed in an undisturbed quiet room with a defined stable temperature (23 ± 1 °C) after a 20-minute rest period in the supine position. The room temperature was monitored and adjusted if necessary using air conditioning, as the outside temperature was usually >25 °C.

Microvascular reactivity was evaluated using an LSCI system with a laser wavelength of 785 nm (PeriCam PSI system, Perimed, Järfälla, Sweden), which enabled us to perform noninvasive and continuous measurements of cutaneous microvascular perfusion changes, which were measured in arbitrary perfusion units (APUs). The images were analyzed using the indicated software (PIMSoft, Perimed, Järfälla, Sweden).

High-frame-rate LSCI is a recently marketed technique based on the analysis of the variations in speckle contrast, which allows measurements of fast changes in skin microvascular blood flow over wide skin areas, with very good interday reproducibility (17). In fact, LSCI measures skin perfusion over wide areas (up to 100 cm²) with a high frequency (up to 100 images/s). LSCI thus has the advantages of good temporal and spatial resolution (22).

One skin site on the ventral surface of the forearm was randomly chosen for the recordings. Hair, broken skin, areas of skin pigmentation and visible veins were avoided, and two drug-delivery electrodes were installed using adhesive discs (LI 611, Perimed, Järfälla, Sweden), as previously described (16, 23). The following three measurement areas were identified: two measurement areas within the electrodes containing acetylcholine (ACh) or sodium nitroprusside (SNP) and another measurement area (baseline control) adjacent to the electrodes. A vacuum cushion (AB Germa, Kristianstad, Sweden) was used to minimize recording artifacts generated by arm movements. ACh (2% w/v) or SNP (Sigma Chemical Co., St. Louis, USA) iontophoresis was performed using a micropharmacology system (PF 751 PeriIont USB Power Supply, Perimed, Sweden) with increasing anodal currents of 30, 60, 90, 120, 150 and 180 μA, which were administered at 10-second intervals spaced 1 minute apart. The total charges for the above currents were 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 mC, respectively. The dispersive electrode was attached approximately 15 cm from the electrophoresis chamber. The pharmacological test results are expressed as peak values representing the maximal vasodilation observed after the highest ACh or SNP doses. Skin blood flow measurements in arbitrary perfusion units (APUs) were divided by mean arterial pressure values to yield cutaneous vascular conductance (CVC) in APU/mmHg. The use of the area under the blood flow/time curve for assessing skin microvascular reactivity using laser technology is well validated because it represents the global flow response to different physiological and pharmacological stimuli (24).

During the postocclusive reactive hyperemia (PORH) test, arterial occlusion was performed with suprasystolic pressure, defined as 50 mmHg above the systolic arterial pressure, by applying a sphygmomanometer for 3 min (25). Following the release of pressure, the maximum flux was measured. The PORH tests were always performed after the iontophoresis of ACh and SNP to avoid interference with these pharmacological tests, which are limited to local responses in the skin. In contrast, the hyperemic response is a global response that extends to the skin of the whole forearm and thus could interfere with subsequent tests of microvascular reactivity.

Statistical analysis

The results are presented as the mean ± SD. Values that did not follow a Gaussian distribution are presented as medians (25^th–75^th percentile) (Shapiro–Wilk normality test). The results were analyzed using two-tailed unpaired Student's t tests, Mann‒Whitney U tests or Fisher's exact tests, when appropriate. P values <0.05 were considered to indicate statistical significance. All the statistical analyses were performed using Prism, version 7.0 (GraphPad Software Inc. La Jolla, CA, USA).

Clinical characteristics of the study subjects

The clinical characteristics of the hypertensive patients are presented in Table 1, and information regarding current cardiovascular drug use is presented in Table 2. Both groups were paired by age (P=1); glycemic profile, including fasting plasma glucose (P=0.49) and glycated hemoglobin (P=0.30); and urinary creatinine (P=0.14). The number of patients with diabetes (P=1), but not that of those with chronic kidney disease (0.05), was also equivalent in both groups. Nonetheless, there were more female patients in the RH + MAU group (P=0.02) than in the RH group. The UACR was 7.6 (5-14.1) mg/g in the RH group and 89.6 (56.5-250.9) mg/g in the RH + MAU group (P<0.0001). Diastolic (P=0.03) and mean (P=0.02) arterial pressures were greater in the RH + MAU group. There were no differences in the UACR between males and females in the RH (P=0.25) or RH-MAU (P=0.75) groups. Finally, LDL-cholesterol plasma levels were greater in the RH + MAU group (P<0.0001).

As expected, the patients took antihypertensive medications from several classes, with a very high proportion of patients taking angiotensin receptor blockers and diuretics followed by calcium channel blockers and direct vasodilators. Statin use was also very frequent (>90%).

Evaluation of skin microvascular flow and reactivity

Systemic microvascular reactivity

The basal cutaneous vascular conductance (CVC) values were 0.33 ± 0.13 and 0.27 ± 0.09 APU/mmHg in the RH and RH + MAU individuals, respectively (P=0.07).

The peak CVCs during postocclusive reactive hyperemia (PORH) were 0.64 ± 0.15 and 0.55 ± 0.15 APU/mmHg in the RH and RH + MAU patients, respectively (P=0.03). The delta values (peak – baseline) during PORH were 0.31 ± 0.11 and 0.25 ± 0.09 APU/mmHg in the RH and RH + MAU patients, respectively (P=0.04) (Figure 1).

Cutaneous ACh iontophoresis induced significant, current-related increases in the microvascular CVC in both the RH and RH + MAU groups of patients (Figure 2). The areas under the curve of vasodilation induced by ACh were similar between the groups: 69.49 ± 6.91 and 67.15 ± 6.91 APU/mmHg in the RH and RH + MAU individuals, respectively (P=0.23).

Cutaneous SNP iontophoresis induced significant, current-related increases in microvascular CVC in both the RH and RH + MAU groups of patients (Figure 3). The areas under the curve of vasodilation induced by SNP were greater in the group without MAU than in the group with MAU: 58.54 ± 6.40 and 53.42 ± 6.40 APU/mmHg in the RH and RH + MAU individuals, respectively (P=0.003).

The main findings of this study are that endothelial-dependent microvascular vasodilation induced by postocclusive reactive hyperemia, but not by iontophoresis of ACh, is decreased in hypertensive patients with MAU compared with age-matched hypertensive patients without MAU. Endothelial-independent microvascular vasodilation induced by iontophoresis of SNP was also reduced in hypertensive patients with MAU.

MAU is currently accepted as a biomarker of renal, cardiac and vascular damage that is predictive of the further development of cardiovascular events, renal failure and diabetes (26) and is also closely associated with the prevalence of arterial hypertension (27). Moreover, MAU has been associated with subclinical hypertensive organ damage, characterized by a higher prevalence of concentric left ventricular hypertrophy and subclinical impairment of left ventricular performance, as well as the presence of carotid atherosclerosis (28, 29).

Notably, nondiabetic MAU is a biomarker of endothelial dysfunction that contributes to both atherosclerotic macrovascular disease and renal microvascular disease (30). Moreover, systemic endothelial dysfunction might explain the prognostic value of MAU for cardiovascular events and end-organ damage (31, 32). Endothelial dysfunction of the renal microcirculation causes MAU by increasing glomerular capillary wall permeability and increasing intraglomerular pressure (33), which leads to glomerular capillary rarefaction—mainly in patients with arterial hypertension (34)—and further increases in intraglomerular pressure (35). In fact, the presence of MAU in patients with arterial hypertension is a strong indicator of microvascular damage (36).

In the present study, hypertensive patients with MAU showed reduced endothelial-dependent systemic microvascular reactivity compared to their counterparts without MAU, as demonstrated by an attenuation of microvascular vasodilation induced by PORH. On the other hand, ACh-induced vasodilation did not differ between groups. Using strain-gauge plethysmography to study forearm vasodilation in response to intrabrachial infusion of acetylcholine, Taddei et al. reported that vascular responses were similar in hypertensive patients with or without MAU (37).

In this context, it is essential to discuss the different mechanisms involved in the physiological and pharmacological tests used to evaluate endothelial function in the present study. Dissimilar endothelial mediators, including nitric oxide (NO), cyclooxygenase metabolites, and endothelium-derived hyperpolarizing factor (EDHF), are involved in the hyperemic response that follows arterial occlusion (PORH, physiological provocation) (22). In fact, large-conductance calcium-activated potassium (BKCa) channels appear to be involved, suggesting the participation of EDHF in this response (38). A variety of endothelium-derived vasodilator mediators, including EDHF, NO, and prostaglandins, also appear to be released following ACh skin iontophoresis (pharmacological provocation) (22, 39-41). However, it has been recently demonstrated that arteries from hypertensive patients present with late-phase acetylcholine-induced endothelium-dependent contraction due to arachidonic acid-derived bioactive products, which is likely related to low-grade, chronic vascular inflammation (42). Thus far, it is unclear whether this process is fundamental for the genesis of hypertension or if it exacerbates vascular tissue injury leading to malignant hypertension (42).

The presence of MAU is also associated with different vascular structural alterations (43, 44), such as increased thickness of the intima and media of the carotid artery compared with patients with normoalbuminuria (45), and predicts the development and progression of carotid atherosclerosis (46). Moreover, in addition to functional and mechanical changes, structural alterations of the microcirculation, mainly represented by microvascular remodeling, are observed in patients with essential hypertension, even at very early stages, contributing to the development of hypertension complications and impacting cardiovascular prognosis (47-49).

The results of the present study also revealed reduced endothelial-independent (SNP-induced) microvascular reactivity in hypertensive patients with MAU compared with patients without MAU, indicating impaired microvascular smooth muscle function. Considering that MAU indicates systemic vascular structural alterations and that SNP is a direct nitric oxide donor that skirts the endothelium and directly relaxes adjacent vascular smooth muscle cells, this result indicates structural alterations in microvessels (arteriolar remodeling) in patients with MAU. In this context, we have already shown that patients diagnosed with early-onset coronary artery disease have structural microvascular alterations concurrent with increased carotid intima-media thickness (18).

In our study, there was a similar percentage (approximately 30%) of diabetic patients in both groups, thus precluding the conclusion that differences in microcirculatory reactivity observed in the RH+MAU group compared to the RH without MAU group could be related to well-known systemic microvascular alteration characteristics of diabetic angiopathy, which is one of the most common complications that arises from chronic diabetes (50).

In our observational study, the RH+MAU group had a greater number of women (almost twofold); nevertheless, the UACR did not differ between males and females in either group, i.e., those who did or did not present with MAU. The fact that we observed a greater number of women with MAU when recruiting an equal number of patients of both sexes is in line with previous studies demonstrating a greater prevalence of women with micro- and macroalbuminuria among individuals over 20 years of age (51).

As expected, the prevalence of chronic kidney disease was greater in the RH+MAU group, as classified according to the creatinine-based Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI₂₀₀₉) (52). In this case, an estimated glomerular filtration rate (eGFR)<60 mL/min/1.73 m²is considered to represent mild renal insufficiency for both men and women. In our study, the mean eGFR did not differ between the two groups.

Both diastolic and mean arterial pressure values were greater in the RH+MAU group than in the RH without MAU group. These results confirm the correlation between arterial pressure and urinary albumin excretion that has already been demonstrated concerning both casual values of arterial pressure (53, 54) and ambulatory blood pressure measurements (55).

Notably, LDL-cholesterol levels were greater in patients with RH+MAU than in those without MAU, despite the absence of any difference in the frequency of statin use between the groups. It is reasonable to assume that the group with worse microvascular function would have higher levels of LDL cholesterol since reduced endothelial function is associated with hypercholesterolemia. A possible explanation is the decreased synthesis of nitric oxide in the vascular endothelium of these individuals. In a previous study, a reduction in endothelium-dependent vasodilation was observed in individuals with hypercholesterolemia, while there was no difference in endothelium-independent vasodilation, indicating that the reduction in vasodilation was not a consequence of a lower smooth muscle response to the vasodilatory stimulus (56).

Study limitations

As this was a cross-sectional observational study, no causal relationships could be identified, only associations; therefore, we were unable to determine whether endothelial dysfunction might have contributed to the development of MAU. Furthermore, although we recruited an equal number of men and women, the RH+MAU group had a greater percentage of women, making the sample unmatched for sex. However, it is worth noting that other studies have also reported a greater prevalence of RH+MAU in women than in men.

In this study, there was evidence of microvascular dysfunction in patients with RH and MAU, with altered endothelium-dependent and endothelium-independent vasodilatation. This may imply a worse prognosis for these patients. These findings may suggest that additional risk stratification strategies for identifying microvascular dysfunction might be useful in patients with RH and that more intensive therapeutic strategies for the control of blood pressure and associated conditions, such as metabolic syndrome, are needed to avoid further damage.

ACh, acetylcholine; APU, arbitrary perfusion units; CVC, cutaneous vascular conductance; eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin; LSCI, laser speckle contrast imaging; MAU, microalbuminuria; SNP, sodium nitroprusside; PORH, postocclusive reactive hyperemia; RH, resistant arterial hypertension; UACR, urine albumin-to-creatinine ratio.

INTRODUCTION

Systemic arterial hypertension (SAH) is the result of complex interactions between environmental, genetic and social factors that initiate and perpetuate blood pressure elevations and is one of the most prevalent cardiovascular diseases worldwide (1, 2). SAH is characterized by important changes in systemic microvascular reactivity and density, as demonstrated by studies reporting altered microvascular endothelial responses to a variety of stimuli in hypertensive patients, identified mostly via laser microvascular perfusion monitoring technology (3–6). Given that microvascular dysfunction and rarefaction are unquestionably involved in the pathophysiology of SAH and that hypertension can lead to microvascular alterations, a bidirectional causality most likely exists between the two conditions (7).

In some cases, SAH can progress to a phenotype called resistant arterial hypertension (RH) (8). RH is defined as either the inability to achieve blood pressure control despite the administration of at least three antihypertensive medications at maximal or maximally tolerated dosages, including a diuretic, or controlled blood pressure requiring at least four medications (9). Typical three-drug regimens include a renin-angiotensin system inhibitor, a calcium channel blocker, and a thiazide or thiazide-like diuretic (9).

Microalbuminuria (MAU) is defined as an abnormally increased excretion rate of albumin in the urine in the range of 30–299 mg/g creatinine and is associated with increased mortality independent of the estimated glomerular filtration rate (eGFR) (10, 11). MAU is associated with different cardiometabolic conditions, including salt sensitivity and hypertension, obesity, insulin resistance and diabetes, as well as other components of metabolic syndrome, such as dyslipidemia (12). Moreover, MAU indicates microvascular injury and is independently associated with a greater risk of major adverse cardiovascular events and all-cause mortality in hypertensive patients, especially in those with RH (13–15).

As a marker of microvessel injury, MAU may be associated with other microvascular damage indicators, such as endothelial dysfunction, as demonstrated by different methods. Laser speckle contrast imaging (LSCI) provides an innovative approach for noninvasively evaluating systemic microvascular endothelial function, with physiological (postocclusive reactive hyperemia) or pharmacological (acetylcholine and sodium nitroprusside) stimulation to evaluate systemic endothelial-dependent and endothelial-independent microvascular reactivity (16). A major advantage of this technique is that LSCI is more reproducible than earlier procedures, such as laser Doppler flowmetry and laser Doppler imaging (17). LSCI has also previously been shown to be an effective noninvasive technique for evaluating systemic microvascular reactivity in patients who present with cardiovascular and metabolic diseases (16, 18).

Therefore, this study aimed to evaluate systemic microvascular function in patients with RH, with or without MAU, to identify possible associations between MAU and endothelial dysfunction in the context of RH. The combination of the two alterations (MAU and endothelial dysfunction) might indicate a greater risk profile, which could merit more intensive therapeutic interventions to improve patient prognosis.

DISCUSSION

The main findings of this study are that endothelial-dependent microvascular vasodilation induced by postocclusive reactive hyperemia, but not by iontophoresis of ACh, is decreased in hypertensive patients with MAU compared with age-matched hypertensive patients without MAU. Endothelial-independent microvascular vasodilation induced by iontophoresis of SNP was also reduced in hypertensive patients with MAU.

MAU is currently accepted as a biomarker of renal, cardiac and vascular damage that is predictive of the further development of cardiovascular events, renal failure and diabetes (26) and is also closely associated with the prevalence of arterial hypertension (27). Moreover, MAU has been associated with subclinical hypertensive organ damage, characterized by a higher prevalence of concentric left ventricular hypertrophy and subclinical impairment of left ventricular performance, as well as the presence of carotid atherosclerosis (28, 29).

Notably, nondiabetic MAU is a biomarker of endothelial dysfunction that contributes to both atherosclerotic macrovascular disease and renal microvascular disease (30). Moreover, systemic endothelial dysfunction might explain the prognostic value of MAU for cardiovascular events and end-organ damage (31, 32). Endothelial dysfunction of the renal microcirculation causes MAU by increasing glomerular capillary wall permeability and increasing intraglomerular pressure (33), which leads to glomerular capillary rarefaction—mainly in patients with arterial hypertension (34)—and further increases in intraglomerular pressure (35). In fact, the presence of MAU in patients with arterial hypertension is a strong indicator of microvascular damage (36).

In the present study, hypertensive patients with MAU showed reduced endothelial-dependent systemic microvascular reactivity compared to their counterparts without MAU, as demonstrated by an attenuation of microvascular vasodilation induced by PORH. On the other hand, ACh-induced vasodilation did not differ between groups. Using strain-gauge plethysmography to study forearm vasodilation in response to intrabrachial infusion of acetylcholine, Taddei et al. reported that vascular responses were similar in hypertensive patients with or without MAU (37).

In this context, it is essential to discuss the different mechanisms involved in the physiological and pharmacological tests used to evaluate endothelial function in the present study. Dissimilar endothelial mediators, including nitric oxide (NO), cyclooxygenase metabolites, and endothelium-derived hyperpolarizing factor (EDHF), are involved in the hyperemic response that follows arterial occlusion (PORH, physiological provocation) (22). In fact, large-conductance calcium-activated potassium (BKCa) channels appear to be involved, suggesting the participation of EDHF in this response (38). A variety of endothelium-derived vasodilator mediators, including EDHF, NO, and prostaglandins, also appear to be released following ACh skin iontophoresis (pharmacological provocation) (22, 39–41). However, it has been recently demonstrated that arteries from hypertensive patients present with late-phase acetylcholine-induced endothelium-dependent contraction due to arachidonic acid-derived bioactive products, which is likely related to low-grade, chronic vascular inflammation (42). Thus far, it is unclear whether this process is fundamental for the genesis of hypertension or if it exacerbates vascular tissue injury leading to malignant hypertension (42).

The presence of MAU is also associated with different vascular structural alterations (43, 44), such as increased thickness of the intima and media of the carotid artery compared with patients with normoalbuminuria (45), and predicts the development and progression of carotid atherosclerosis (46). Moreover, in addition to functional and mechanical changes, structural alterations of the microcirculation, mainly represented by microvascular remodeling, are observed in patients with essential hypertension, even at very early stages, contributing to the development of hypertension complications and impacting cardiovascular prognosis (47–49).

The results of the present study also revealed reduced endothelial-independent (SNP-induced) microvascular reactivity in hypertensive patients with MAU compared with patients without MAU, indicating impaired microvascular smooth muscle function. Considering that MAU indicates systemic vascular structural alterations and that SNP is a direct nitric oxide donor that skirts the endothelium and directly relaxes adjacent vascular smooth muscle cells, this result indicates structural alterations in microvessels (arteriolar remodeling) in patients with MAU. In this context, we have already shown that patients diagnosed with early-onset coronary artery disease have structural microvascular alterations concurrent with increased carotid intima-media thickness (18).

In our study, there was a similar percentage (approximately 30%) of diabetic patients in both groups, thus precluding the conclusion that differences in microcirculatory reactivity observed in the RH + MAU group compared to the RH without MAU group could be related to well-known systemic microvascular alteration characteristics of diabetic angiopathy, which is one of the most common complications that arises from chronic diabetes (50).

In our observational study, the RH + MAU group had a greater number of women (almost twofold); nevertheless, the UACR did not differ between males and females in either group, i.e., those who did or did not present with MAU. The fact that we observed a greater number of women with MAU when recruiting an equal number of patients of both sexes is in line with previous studies demonstrating a greater prevalence of women with micro- and macroalbuminuria among individuals over 20 years of age (51).

As expected, the prevalence of chronic kidney disease was greater in the RH + MAU group, as classified according to the creatinine-based Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI₂₀₀₉) (52). In this case, an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m² is considered to represent mild renal insufficiency for both men and women. In our study, the mean eGFR did not differ between the two groups.

Both diastolic and mean arterial pressure values were greater in the RH + MAU group than in the RH without MAU group. These results confirm the correlation between arterial pressure and urinary albumin excretion that has already been demonstrated concerning both casual values of arterial pressure (53, 54) and ambulatory blood pressure measurements (55).

Notably, LDL-cholesterol levels were greater in patients with RH + MAU than in those without MAU, despite the absence of any difference in the frequency of statin use between the groups. It is reasonable to assume that the group with worse microvascular function would have higher levels of LDL cholesterol since reduced endothelial function is associated with hypercholesterolemia. A possible explanation is the decreased synthesis of nitric oxide in the vascular endothelium of these individuals. In a previous study, a reduction in endothelium-dependent vasodilation was observed in individuals with hypercholesterolemia, while there was no difference in endothelium-independent vasodilation, indicating that the reduction in vasodilation was not a consequence of a lower smooth muscle response to the vasodilatory stimulus (56).

Carey RM, Muntner P, Bosworth HB, Whelton PK. Prevention and Control of Hypertension: JACC Health Promotion Series. J Am Coll Cardiol. 2018;72(11):1278–93.
Carey RM, Wright JT, Jr., Taler SJ, Whelton PK. Guideline-Driven Management of Hypertension: An Evidence-Based Update. Circ Res. 2021;128(7):827–46.
Farkas K, Kolossvary E, Jarai Z, Nemcsik J, Farsang C. Non-invasive assessment of microvascular endothelial function by laser Doppler flowmetry in patients with essential hypertension. Atherosclerosis. 2004;173(1):97–102.
Farkas K, Nemcsik J, Kolossvary E, Jarai Z, Nadory E, Farsang C, et al. Impairment of skin microvascular reactivity in hypertension and uraemia. Nephrol Dial Transplant. 2005;20(9):1821–7.
Cupisti A, Rossi M, Placidi S, Fabbri A, Morelli E, Vagheggini G, et al. Responses of the skin microcirculation to acetylcholine in patients with essential hypertension and in normotensive patients with chronic renal failure. Nephron. 2000;85(2):114–9.
Kaiser SE, Sanjuliani AF, Estato V, Gomes MB, Tibirica E. Antihypertensive treatment improves microvascular rarefaction and reactivity in low-risk hypertensive individuals. Microcirculation. 2013;20(8):703–16.
Yannoutsos A, Levy BI, Safar ME, Slama G, Blacher J. Pathophysiology of hypertension: interactions between macro and microvascular alterations through endothelial dysfunction. J Hypertens. 2014;32(2):216–24.
Chan RJ, Helmeczi W, Hiremath SS. Revisiting resistant hypertension: a comprehensive review. Intern Med J. 2023;53(10):1739–51.
Filippone EJ, Naccarelli GV, Foy AJ. Controversies in Hypertension V: Resistant and Refractory Hypertension. Am J Med. 2024;137(1):12–22.
O'Hare AM, Hailpern SM, Pavkov ME, Rios-Burrows N, Gupta I, Maynard C, et al. Prognostic implications of the urinary albumin to creatinine ratio in veterans of different ages with diabetes. Arch Intern Med. 2010;170(11):930–6.
Chang DR, Yeh HC, Ting IW, Lin CY, Huang HC, Chiang HY, et al. The ratio and difference of urine protein-to-creatinine ratio and albumin-to-creatinine ratio facilitate risk prediction of all-cause mortality. Sci Rep. 2021;11(1):7851.
Thomas G, Sehgal AR, Kashyap SR, Srinivas TR, Kirwan JP, Navaneethan SD. Metabolic syndrome and kidney disease: a systematic review and meta-analysis. Clin J Am Soc Nephrol. 2011;6(10):2364–73.
Hong Z, Jiang Y, Liu P, Zhang L. Association of microalbuminuria and adverse outcomes in hypertensive patients: a meta-analysis. Int Urol Nephrol. 2021;53(11):2311–9.
Oliveras A, Armario P, Sierra C, Arroyo JA, Hernandez-del-Rey R, Vazquez S, et al. Urinary albumin excretion at follow-up predicts cardiovascular outcomes in subjects with resistant hypertension. Am J Hypertens. 2013;26(9):1148–54.
Xia F, Liu G, Shi Y, Zhang Y. Impact of microalbuminuria on incident coronary heart disease, cardiovascular and all-cause mortality: a meta-analysis of prospective studies. Int J Clin Exp Med. 2015;8(1):1–9.
Cordovil I, Huguenin G, Rosa G, Bello A, Kohler O, de Moraes R, et al. Evaluation of systemic microvascular endothelial function using laser speckle contrast imaging. Microvasc Res. 2012;83(3):376–9.
Roustit M, Millet C, Blaise S, Dufournet B, Cracowski JL. Excellent reproducibility of laser speckle contrast imaging to assess skin microvascular reactivity. Microvasc Res. 2010;80(3):505–11.
Souza EG, De Lorenzo A, Huguenin G, Oliveira GM, Tibirica E. Impairment of systemic microvascular endothelial and smooth muscle function in individuals with early-onset coronary artery disease: studies with laser speckle contrast imaging. Coron Artery Dis. 2014;25(1):23–8.
Stevens PE, Levin A, Kidney Disease: Improving Global Outcomes Chronic Kidney Disease Guideline Development Work Group M. Evaluation and management of chronic kidney disease: synopsis of the kidney disease: improving global outcomes 2012 clinical practice guideline. Ann Intern Med. 2013;158(11):825–30.
Toto RD. Microalbuminuria: definition, detection, and clinical significance. J Clin Hypertens (Greenwich). 2004;6(11 Suppl 3):2–7.
Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF, 3rd, Feldman HI, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009;150(9):604–12.
Roustit M, Cracowski JL. Assessment of endothelial and neurovascular function in human skin microcirculation. Trends Pharmacol Sci. 2013;34(7):373–84.
Escobar S, Pecanha D, Duque M, Duque A, Crahim V, De Lorenzo A, et al. Evaluation of systemic endothelial-dependent and endothelial-independent microvascular reactivity in metabolically healthy obesity: An observational study. Microvasc Res. 2023;148:104553.
Cracowski JL, Roustit M. Current Methods to Assess Human Cutaneous Blood Flow: An Updated Focus on Laser-Based-Techniques. Microcirculation. 2016;23(5):337–44.
Yvonne-Tee GB, Rasool AH, Halim AS, Wong AR, Rahman AR. Method optimization on the use of postocclusive hyperemia model to assess microvascular function. Clin Hemorheol Microcirc. 2008;38(2):119–33.
Amann K, Wanner C, Ritz E. Cross-talk between the kidney and the cardiovascular system. J Am Soc Nephrol. 2006;17(8):2112–9.
Cerasola G, Cottone S, Mule G, Nardi E, Mangano MT, Andronico G, et al. Microalbuminuria, renal dysfunction and cardiovascular complication in essential hypertension. J Hypertens. 1996;14(7):915–20.
Pontremoli R, Leoncini G, Ravera M, Viazzi F, Vettoretti S, Ratto E, et al. Microalbuminuria, cardiovascular, and renal risk in primary hypertension. J Am Soc Nephrol. 2002;13 Suppl 3:S169-72.
Sierra C, de la Sierra A. Early detection and management of the high-risk patient with elevated blood pressure. Vasc Health Risk Manag. 2008;4(2):289–96.
Pedrinelli R, Dell'Omo G, Penno G, Mariani M. Non-diabetic microalbuminuria, endothelial dysfunction and cardiovascular disease. Vasc Med. 2001;6(4):257–64.
Ochodnicky P, Henning RH, van Dokkum RP, de Zeeuw D. Microalbuminuria and endothelial dysfunction: emerging targets for primary prevention of end-organ damage. J Cardiovasc Pharmacol. 2006;47 Suppl 2:S151-62; discussion S72-6.
Weir MR. Microalbuminuria and cardiovascular disease. Clin J Am Soc Nephrol. 2007;2(3):581–90.
Stehouwer CD, Smulders YM. Microalbuminuria and risk for cardiovascular disease: Analysis of potential mechanisms. J Am Soc Nephrol. 2006;17(8):2106–11.
Triantafyllou A, Anyfanti P, Zabulis X, Gavriilaki E, Karamaounas P, Gkaliagkousi E, et al. Accumulation of microvascular target organ damage in newly diagnosed hypertensive patients. J Am Soc Hypertens. 2014;8(8):542–9.
Brenner BM, Meyer TW, Hostetter TH. Dietary protein intake and the progressive nature of kidney disease: the role of hemodynamically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in aging, renal ablation, and intrinsic renal disease. N Engl J Med. 1982;307(11):652–9.
Ayerden Ebinc F, Haksun E, Ulver DB, Koc E, Erten Y, Reis Altok K, et al. The relationship between vascular endothelial growth factor (VEGF) and microalbuminuria in patients with essential hypertension. Intern Med. 2008;47(17):1511–6.
Taddei S, Virdis A, Mattei P, Ghiadoni L, Sudano I, Arrighi P, et al. Lack of correlation between microalbuminuria and endothelial function in essential hypertensive patients. J Hypertens. 1995;13(9):1003–8.
Lorenzo S, Minson CT. Human cutaneous reactive hyperaemia: role of BKCa channels and sensory nerves. J Physiol. 2007;585(Pt 1):295–303.
Durand S, Tartas M, Bouye P, Koitka A, Saumet JL, Abraham P. Prostaglandins participate in the late phase of the vascular response to acetylcholine iontophoresis in humans. J Physiol. 2004;561(Pt 3):811–9.
Holowatz LA, Thompson CS, Minson CT, Kenney WL. Mechanisms of acetylcholine-mediated vasodilatation in young and aged human skin. J Physiol. 2005;563(Pt 3):965–73.
Noon JP, Walker BR, Hand MF, Webb DJ. Studies with iontophoretic administration of drugs to human dermal vessels in vivo: cholinergic vasodilatation is mediated by dilator prostanoids rather than nitric oxide. Br J Clin Pharmacol. 1998;45(6):545–50.
Edwards JM, McCarthy CG, Wenceslau CF. The Obligatory Role of the Acetylcholine-Induced Endothelium-Dependent Contraction in Hypertension: Can Arachidonic Acid Resolve this Inflammation? Curr Pharm Des. 2020;26(30):3723–32.
Pathak SR, Bhattarai N, Baskota D, Koju RP, Humagain S. Prevalence of Microalbuminuria in Patients of Essential Hypertension and its Correlation with Left Ventricular Hypertrophy and Carotid Artery Intima–media Thickness. Kathmandu Univ Med J (KUMJ). 2022;20(80):417–21.
Rodondi N, Yerly P, Gabriel A, Riesen WF, Burnier M, Paccaud F, et al. Microalbuminuria, but not cystatin C, is associated with carotid atherosclerosis in middle-aged adults. Nephrol Dial Transplant. 2007;22(4):1107–14.
Geraci G, Mule G, Costanza G, Mogavero M, Geraci C, Cottone S. Relationship Between Carotid Atherosclerosis and Pulse Pressure with Renal Hemodynamics in Hypertensive Patients. Am J Hypertens. 2016;29(4):519–27.
Jorgensen L, Jenssen T, Johnsen SH, Mathiesen EB, Heuch I, Joakimsen O, et al. Albuminuria as risk factor for initiation and progression of carotid atherosclerosis in non-diabetic persons: the Tromso Study. Eur Heart J. 2007;28(3):363–9.
Agabiti-Rosei E, Rizzoni D. Microvascular structure as a prognostically relevant endpoint. J Hypertens. 2017;35(5):914–21.
Rizzoni D, Agabiti-Rosei C, De Ciuceis C. State of the Art Review: Vascular Remodeling in Hypertension. Am J Hypertens. 2023;36(1):1–13.
Rizzoni D, Agabiti-Rosei C, Boari GEM, Muiesan ML, De Ciuceis C. Microcirculation in Hypertension: A Therapeutic Target to Prevent Cardiovascular Disease? J Clin Med. 2023;12(15).
Strain WD, Paldanius PM. Diabetes, cardiovascular disease and the microcirculation. Cardiovasc Diabetol. 2018;17(1):57.
Coresh J, Byrd-Holt D, Astor BC, Briggs JP, Eggers PW, Lacher DA, et al. Chronic kidney disease awareness, prevalence, and trends among U.S. adults, 1999 to 2000. J Am Soc Nephrol. 2005; 16: 180–188.
Delanaye P, Cavalier E, Pottel H, Stehle T. New and old GFR equations: a European perspective. Clin Kidney J. 2023;16(9):1375–83.
Pedrinelli R, Giampietro O, Carmassi F, Melillo E, Dell'Omo G, Catapano G, et al. Microalbuminuria and endothelial dysfunction in essential hypertension. Lancet. 1994;344(8914):14–8.
Redon J, Liao Y, Lozano JV, Miralles A, Baldo E, Cooper RS. Factors related to the presence of microalbuminuria in essential hypertension. Am J Hypertens. 1994;7(9 Pt 1):801–7.
Bianchi S, Bigazzi R, Valtriani C, Chiapponi I, Sgherri G, Baldari G, et al. Elevated serum insulin levels in patients with essential hypertension and microalbuminuria. Hypertension. 1994;23(6 Pt 1):681–7.
Casino PR, Kilcoyne CM, Quyyumi AA, Hoeg JM, Panza JA. The role of nitric oxide in endothelium-dependent vasodilation of hypercholesterolemic patients. Circulation. 1993; 88: 2541–2547.

TABLE 1: The clinical characteristics of resistant hypertensive (RH) patients presenting with or without microalbuminuria (MAU).

Characteristics		RH (n=32)	RH + MAU (n=32)	P value
Age (years)	59.1 ± 8.4		59.1 ± 7.7	1
Male, n (%)	21 (66)		11 (34.4)	0.02
Smokers, n (%)	0 (0)		2 (6.25)	0.49
Diabetes status, n (%)	10 (31.2)		9 (28.1)	1
Chronic kidney disease status, n (%)	5 (15.6)		13 (40.6)	0.05
Weight (kg)	89.04 ± 17.9		86.3 ± 15.4	0.53
Height (cm)	167 ± 0.1		164 ± 0.1	0.30
Body mass index (kg/m²)	31.8 ± 5.5		31.9 ± 4.5	0.95
Systolic blood pressure (mmHg)	155 ± 22.5		167 ± 26.8	0.06
Diastolic blood pressure (mmHg)	87.2 ± 14.5		95.5 ± 15.4	0.03
Mean blood pressure (mmHg)	109.8 ± 15.6		119.4 ± 17.7	0.02
Heart rate (bpm)	65.7 ± 11.8		65.8 ± 12.3	0.97
ALT IU/L	20 (14.9-29.5)		17 (13.4-20.7)	0.31
Plasma creatinine (mg/dl)	0.97 (0.86-1.15)		1 (0.79-1.41)	0.87
Urinary creatinine (mg/dl)	116.8 (70.4-167.9)		62.19 (48.2-89.1)	0.14
Urea (mg/dl)	31.5 (29-48.5)		38.3 (30.2-58.7)	0.06
Uric acid (mg/dl)	6.25 ± 1.7		5.7 ± 1.4	0.20
Total cholesterol (mg/dL)	171.3 (141.3-206.9)		162.5 (132.8-187.7)	0.55
Triglycerides (mg/dL)	144 (84-194.5)		156.5 (105.3-200.3)	0.40
HDL-C (mg/dL)	41 (35.1-49.6)		47.3 (39-56.7)	0.08
LDL-C (mg/dL)	109.9 (75-133)		162.5 (132.8-187.7)	<0.0001
Fasting glucose (mg/dL)	106 (94-130)		104 (89-116)	0.49
HbA1c (%)	6 (5.2-6.5)		6 (5.7-6.8)	0.30
eGFR (mL/min/1.73m²)	77.8 ± 21.1		71.5 ± 23.1	0.27
UACR (mg/g)	7.6 (5-14.1)		89.6 (56.5-250.9)	<0.0001
Male UACR (mg/g)	9.2 (5.1-15.8)		74.1 (56.9-410.4)	<0.0001
Female UACR (mg/g)	5.8 (5-8.7)		116.3 (55.8-250.8)	<0.0001

The results are presented as the mean ± SEM. For values that did not follow a Gaussian distribution, the medians (25^th–75^th percentile) are presented (Shapiro–Wilk normality test).

Abbreviations: ALT, alanine transaminase; ACE, angiotensin-converting enzyme; eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin; HDL-C, high-density lipoprotein; LDL-C, low-density lipoprotein; UACR, urine albumin-to-creatinine ratio.

P values were estimated using two-tailed unpaired Student's t tests, Mann‒Whitney tests or Fisher's exact tests, as appropriate. Values in bold represent statistically significant differences between groups.

TABLE 2: Cardiovascular drugs used for the treatment of resistant hypertensive (RH) patients presenting with or without microalbuminuria (MAU).

Usual medications	RH		RH + MAU	P value
Angiotensin receptor blockers, n (%)		30 (94)	29 (91)	1
ACE inhibitors, n (%)		2 (6)	2 (6)	1
Calcium channel blockers, n (%)		25 (78)	27 (84)	0.75
Diuretics		32 (100)	32 (100)	1
Potassium-sparing diuretics, n (%)		20 (62)	21 (65)	1
Central α₂-agonists, n (%)		18 (56)	21 (65)	0.61
Beta blockers, n (%)		22 (69)	24 (75)	0.78
Direct vasodilators, n (%)		28 (87)	25 (78)	0.51
Statins, n (%)		31 (97)	31 (97)	1

Abbreviations: ACE, angiotensin-converting enzyme.

P values were estimated using the chi-square test (Fisher's exact test).

There is NO conflict of interest to disclose.

Reduced Systemic Microvascular Function in Patients With Resistant Hypertension and Microalbuminuria: an Observational Study

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