journal article
LitStream Collection
The Effects of Dapagliflozin on Systemic and Renal Vascular Function Display an Epigenetic Signature
Solini,, Anna;Seghieri,, Marta;Giannini,, Livia;Biancalana,, Edoardo;Parolini,, Federico;Rossi,, Chiara;Dardano,, Angela;Taddei,, Stefano;Ghiadoni,, Lorenzo;Bruno, Rosa, Maria
doi: 10.1210/jc.2019-00706pmid: 31162549
Abstract Context Mechanisms mediating the cardiovascular and renal protection exerted by SGLT2 inhibitors are still partially unknown. We investigated whether dapagliflozin modulates systemic and renal vascular function and structure, and induces epigenetic modifications. Subjects and Methods Forty hypertensive patients with type 2 diabetes were randomly assigned to 4-week treatment with dapagliflozin 10 mg or hydrochlorothiazide (HCT) 12.5 mg. Routine analyses; plasma renin activity; aldosterone, catecholamine, and 24-hour urinary electrolyte levels; flow-mediated dilation (FMD) of the brachial artery; carotid-femoral pulse-wave velocity (PWV); augmentation index; and resistive index and dynamic renal resistive index (DRIN) were measured at baseline and after treatment. Circulating miRNAs (miRs) related to heart failure (miR30e-5p, miR199a-3p), endothelial dysfunction (miR27b and miR200b), and renal function (miR130b-3p, miR21-5p) were assessed and related to the effects of treatments. Results Dapagliflozin and HCT marginally lowered blood pressure. Fasting glucose was lowered, whereas 24-hour diuresis, glycosuria, and osmolar clearance were increased by dapagliflozin (P < 0.001 for all), without affecting sodium excretion and glomerular filtration rate. Magnesium levels significantly increased after dapagliflozin treatment (P = 0.02). Neither dapagliflozin nor HCT modified FMD or PWV. DRIN did not vary in the dapagliflozin group, whereas it increased in the HCT group (P = 0.047 for time by treatment interaction). Both treatments induced variations in the expression of some miRs; dapagliflozin, but not HCT, significantly up-regulated miR30e-5p and downregulated miR199a-3p. Conclusion A putative epigenetic regulation of the protecting cardiovascular effect exerted by SGLT2 inhibitors was found. Dapagliflozin might exert nephroprotection by preserving renal vasodilating capacity. SGLT2 inhibition confers cardiovascular (CV) and renal protection that goes beyond what is expected by improvement in glycemic control. It has been suggested that hemodynamic changes related to plasma volume depletion are the prevalent mechanisms for reduction of CV death with empagliflozin treatment compared with placebo (1). A similar mode of action, driven primarily by plasma volume and hemodynamic variations, has been shown in patients treated with canagliflozin (2, 3). Several real-life studies and meta-analyses (4–6), as well as the recent results of the DECLARE-TIMI 58 trial (7), indicate that dapagliflozin also exerts CV protection, suggesting that a class effect indeed might exist. Studies have shown an amelioration of vascular properties in subjects with type 2 diabetes (T2D), either in early-stage disease (8) or with longer duration of disease (9), who received dapagliflozin, possibly contributing to improvement in CV outcomes. However, it is less clear whether these vascular changes are due to intrinsic properties of dapagliflozin or are a consequence of the blood pressure (BP)-lowering effect, plasma volume contraction, and related neurohumoral changes. Recently, we demonstrated that compared with hydrochlorothiazide (HCT) treatment in patients with T2D, an acute treatment (i.e., 2 days) with dapagliflozin improved systemic and renal vascular stiffness and endothelial dysfunction independently of glucose levels, BP, and natriuresis (10). We also hypothesized that a reduction in oxidative stress could contribute to such beneficial effects. MicroRNAs (miRs), noncoding RNA molecules modulating intracellular processes, are being proposed as potential molecules for the prediction of therapeutic response (11). The expression of some miRs enables tracking of hemodynamic status in heart failure (12, 13), and thus is relevant to the use of SGLT2 inhibitors, which are also protective in the setting of heart failure. Moreover, miRs modulating endothelial function and atherosclerosis (14–17), as well as those involved in inflammatory and fibrotic processes in kidney disease (18–20), might play a role in underlying vascular protection induced by SGLT2 inhibitors. Following the design of our pilot study (10), in the current prospective, randomized, open-label, blinded end point clinical trial, we compared a 4-week treatment with dapagliflozin with that of HCT in patients with hypertension and T2D. Our aims were (1) to provide an extensive characterization of the effect of dapagliflozin on endothelial function and systemic and renal vascular function, as assessed by gold-standard noninvasive measurements, and also in relation to changes in sodium (Na) and volume status, the renin-angiotensin system (RAS), neurohormones, and BP values; and (2) to combine such detailed vascular phenotyping with the evaluation of an epigenetic signature marking the vascular effects of SGLT2 inhibitors by testing whether the baseline expression of some miRs could track the clinical effects of dapagliflozin (thus serving as a useful predictor of vascular-related outcomes) and whether dapagliflozin might modify such epigenetic trait. Methods Subjects Forty patients with hypertension and T2D, consecutively recruited among those referred to the diabetes and metabolic diseases outpatient clinic at the University Hospital of Pisa in 2017, participated in this phase IV, open-label, head-to-head study. Inclusion and exclusion criteria are listed in Table 1. Background medication was unchanged during the study. No other antihypertensive drugs in addition to angiotensin-converting enzyme inhibitors were allowed. Similarly, no medications or substances potentially able to interfere with BP or vascular variables were permitted, except in case of patients receiving stable therapy and maintained at a constant dose for the total study period. The nature and purpose of the study were explained to all subjects before they provided their written consent to participate. The protocol was approved by the local ethics committee and registered in the Registry of the Italian Drug Agency (Agenzia Italiana del Farmaco no. 772/2015). Table 1. Patient Inclusion and Exclusion Criteria Inclusion Criteria Exclusion Criteria Age 40–75 y Clinical BP >160/100 mm Hg Both sexes A moderate to severe renal function impairment (eGFR <60 mL/min/1.73 m2) BMI <40 kg/m2 Severe hepatic insufficiency T2D stably treated with any other oral antihyperglycemic drug than SGLT2 inhibitors SGLT2 inhibitors or insulin treatment within 4 weeks immediately preceding the study HbA1c <64 mmol/mol Volume depletion or potential risk for dehydration (e.g., use of other diuretics, laxatives, chronic diarrhea) Clinical BP values not at target (BP >130/80 mm Hg) despite a therapeutic dose of ACE inhibitors Acute coronary syndrome or hospitalization for unstable angina or acute myocardial infarction within 2 mo prior to enrolment Ability to comply with the study protocol Acute stroke or transient ischemic attack within 2 mo prior to enrolment Signed informed consent before any trial related activity Moderate to severe congestive heart failure defined as New York Heart Association class III or greater, unstable or acute congestive heart failure Malignancies or hematologic disorders Any other medical condition that would interfere with the trial, according to the investigator's judgment Inclusion Criteria Exclusion Criteria Age 40–75 y Clinical BP >160/100 mm Hg Both sexes A moderate to severe renal function impairment (eGFR <60 mL/min/1.73 m2) BMI <40 kg/m2 Severe hepatic insufficiency T2D stably treated with any other oral antihyperglycemic drug than SGLT2 inhibitors SGLT2 inhibitors or insulin treatment within 4 weeks immediately preceding the study HbA1c <64 mmol/mol Volume depletion or potential risk for dehydration (e.g., use of other diuretics, laxatives, chronic diarrhea) Clinical BP values not at target (BP >130/80 mm Hg) despite a therapeutic dose of ACE inhibitors Acute coronary syndrome or hospitalization for unstable angina or acute myocardial infarction within 2 mo prior to enrolment Ability to comply with the study protocol Acute stroke or transient ischemic attack within 2 mo prior to enrolment Signed informed consent before any trial related activity Moderate to severe congestive heart failure defined as New York Heart Association class III or greater, unstable or acute congestive heart failure Malignancies or hematologic disorders Any other medical condition that would interfere with the trial, according to the investigator's judgment Abbreviations: ACE, angiotensin-converting enzyme; BMI, body mass index; eGFR, estimated glomerular filtration rate. Open in new tab Table 1. Patient Inclusion and Exclusion Criteria Inclusion Criteria Exclusion Criteria Age 40–75 y Clinical BP >160/100 mm Hg Both sexes A moderate to severe renal function impairment (eGFR <60 mL/min/1.73 m2) BMI <40 kg/m2 Severe hepatic insufficiency T2D stably treated with any other oral antihyperglycemic drug than SGLT2 inhibitors SGLT2 inhibitors or insulin treatment within 4 weeks immediately preceding the study HbA1c <64 mmol/mol Volume depletion or potential risk for dehydration (e.g., use of other diuretics, laxatives, chronic diarrhea) Clinical BP values not at target (BP >130/80 mm Hg) despite a therapeutic dose of ACE inhibitors Acute coronary syndrome or hospitalization for unstable angina or acute myocardial infarction within 2 mo prior to enrolment Ability to comply with the study protocol Acute stroke or transient ischemic attack within 2 mo prior to enrolment Signed informed consent before any trial related activity Moderate to severe congestive heart failure defined as New York Heart Association class III or greater, unstable or acute congestive heart failure Malignancies or hematologic disorders Any other medical condition that would interfere with the trial, according to the investigator's judgment Inclusion Criteria Exclusion Criteria Age 40–75 y Clinical BP >160/100 mm Hg Both sexes A moderate to severe renal function impairment (eGFR <60 mL/min/1.73 m2) BMI <40 kg/m2 Severe hepatic insufficiency T2D stably treated with any other oral antihyperglycemic drug than SGLT2 inhibitors SGLT2 inhibitors or insulin treatment within 4 weeks immediately preceding the study HbA1c <64 mmol/mol Volume depletion or potential risk for dehydration (e.g., use of other diuretics, laxatives, chronic diarrhea) Clinical BP values not at target (BP >130/80 mm Hg) despite a therapeutic dose of ACE inhibitors Acute coronary syndrome or hospitalization for unstable angina or acute myocardial infarction within 2 mo prior to enrolment Ability to comply with the study protocol Acute stroke or transient ischemic attack within 2 mo prior to enrolment Signed informed consent before any trial related activity Moderate to severe congestive heart failure defined as New York Heart Association class III or greater, unstable or acute congestive heart failure Malignancies or hematologic disorders Any other medical condition that would interfere with the trial, according to the investigator's judgment Abbreviations: ACE, angiotensin-converting enzyme; BMI, body mass index; eGFR, estimated glomerular filtration rate. Open in new tab Randomization and study design During the screening visit (1 week before study initiation), patients were randomly assigned to receive treatment with once-daily dapagliflozin 10 mg or HCT 12.5 mg as add on to the background therapy with angiotensin-converting enzyme inhibitors. Participants were advised to keep a stable daily Na intake (92 mmol) from the week preceding the study throughout the entire experimental period. Each participant was studied twice as an outpatient: at baseline (visit 0) and after 4 weeks of treatment with dapagliflozin or HCT (visit 1). The randomization list was in a balanced-block design and prepared using a validated system that automates the random assignment of treatment groups to randomization numbers. Patients were sequentially assigned the next study number as they began the study, starting from the lowest number. Visits at the outpatient clinic took place in the morning, with participants having fasted. BP and heart rate were measured at each visit. Blood and urine samples were collected for routine analyses; determinations of plasma renin activity; and measurement of aldosterone, norepinephrine, epinephrine, and 24-hour urinary electrolyte levels at visits 0 and 1; an extra blood sample was taken to measure miR expression. A complete noninvasive vascular evaluation, including flow-mediated dilation (FMD) of the brachial artery, baseline and dynamic renal resistive index (RI), carotid-femoral pulse wave velocity (PWV), and augmentation index (AIx) was also performed. All vascular tests were carried out by the same physician, who has long experience in this field (R.M.B.). All data were analyzed with the data analysts blinded to the treatment arm. Endothelium-dependent and -independent vasodilation in the brachial artery Endothelium-dependent response was assessed by FMD at the brachial artery level, as previously reported (21), using a high-resolution ultrasound machine equipped with 10 MHz linear explorer (MyLab 25; ESAOTE Florence, Italy). After a 1-minute baseline recording, the cuff was inflated for 5 minutes at 300 ± 30 mm Hg around the forearm and then deflated to induce reactive hyperemia. Endothelium-independent vasodilation was obtained after sublingual administration of 25 μg of glyceryl trinitrate (GTN). Brachial artery diameter and flow velocity were continuously monitored by a computerized edge-detection system (Cardiovascular Suite; Quipu, Italy). FMD and response to GTN were calculated as the maximal percentage increase in diameter above baseline. Hyperemic stimulus was quantified by baseline and hyperemic shear rate (shear rate = 8 × mean flow velocity/brachial artery diameter) and hyperemic shear rate area under the curve. The intrasession (1 hour apart) and intersession (30 days apart) coefficients of variation were 7.6% (range, 0.3% to 10.9%) and 11.6% (2.1% to 13.2%) in a group of 20 healthy volunteers (22). Renal RI Three velocimetric measurements of the interlobar renal arteries in the mesorenal area of each kidney were taken with a translumbar or anterior ultrasonographic approach. Renal RI was calculated in both kidneys according to the following formula: (systolic peak velocity − end diastolic velocity)/systolic peak velocity, and was assessed at baseline and 5 minutes after administration of GTN 25 μg. Dynamic renal resistive index (DRIN) was calculated as (postGTN RI − baseline RI) × 100/baseline RI (10) and expressed as %. Arterial tonometry Arterial tonometry (SphygmoCor; AtCor Medical, Sydney, Australia) was performed according to international recommendations (23). Central BP was derived from radial pressure waveform by a validated transfer function and averaged on three measurements. Augmented pressure was calculated as the difference between the second and the first systolic peak, and AIx was calculated as the ratio of augmented pressure to pulse pressure and normalized at a heart rate of 75 bpm. Time to reflection was defined as the total travel time of the pulse wave to the periphery and its return. Carotid-femoral PWV was calculated as the ratio of the surface distance between the two recording sites (direct distance × 0.8) and wave transit time. Three consecutive measurements were recorded and the median value was considered. Biochemistry HbA1c was measured by HPLC using Diabetes Control and Complications Trial–aligned methods. Total and high-density lipoprotein cholesterol were determined by enzymatic methods and low-density lipoprotein cholesterol was calculated using the Friedewald formula. Standard methods were used to measure the other variables. Humoral parameters of the RAS and neurohormonal system Plasma renin activity and aldosterone were sampled with the patient in the supine position after lying down for 15 minutes and were assayed by RIA (DiaSorin, Saluggia, Italy). Plasma norepinephrine and epinephrine were assayed by HPLC. miR expression Circulating miRs were isolated using the robotic workstation QIACUBE (Qiagen, Hilden, Germany) loaded with miRNeasy Serum/Plasma Kit (catalog no. 217184; Qiagen). After an equilibration period at room temperature and a centrifugation step to remove cryoprecipitate, 200 µL of thawed serum was processed following the manufacturer’s instructions. For each patient, 2 µL of sample eluent from the miR isolation procedure was used to prepare cDNA templates using TaqMan Advanced miRNA cDNA Synthesis kit (catalog no. A28007; Applied Biosystems, Foster City, CA). The expression level of miRs was determined using TaqMan Advanced MicroRNA Assays (catalog no. A25576; Applied Biosystems); PCR reactions were run in triplicate using 3 to 5 µL of cDNA template diluted 1:10, and miR levels were expressed as the comparative threshold cycle using two reference miRs (miR-484 and miR-191-5p) selected on the basis of the scientific literature and checking their low variability in our samples. The following miRs were determined in a fasting state at baseline and at the end of the study: miR-30e-5p, miR-199a-3p, miR-27b, miR-200b, miR-130b-3p, miR-27a-3p, and miR-21-5p. Statistical analysis Data are reported as mean ± SD, or as median (interquartile range) for variables with a skewed distribution. Groups were compared by two-sided paired Student t test for normally distributed continuous variables, Wilcoxon rank-sum test for not normally distributed continuous variables, or χ2 for categorical variables, as appropriate. Time series were analyzed by ANOVA for repeated measurements. Group differences over time series were analyzed by two-way ANOVA for repeated measurements; differences between groups were calculated by Tukey honestly significant difference post hoc comparison test. Bonferroni correction for multiple testing was also applied. Relationships between variables were assessed using Spearman correlation analysis. Log transformation was applied for not normally distributed variables when performing two-way ANOVA or Spearman correlation analysis. Statistical analysis was performed using JMP®7.0. A P value ≤ 0.05 was considered statistically significant. Because this was a proof-of-concept study with no previous data available on the effect of dapagliflozin on miRs, a formal sample-size calculation was not possible. However, a post hoc sample-size calculation was performed considering miR-30e-5p as an outcome variable and using data collected in this study. A sample size of 40 patients (n = 20 in each group) was adequate to demonstrate a significant time by group interaction with a power of 0.99 and α of 0.05. Results All patients completed the protocol, and no adverse effects related to the drugs were detected. As shown in Table 2, bio-anthropometric characteristics, including sex, age, and body mass index, were well matched between groups. After 4 weeks, neither dapagliflozin nor HCT significantly modified BP measured on clinic visits. Whereas HbA1c was similar between groups, fasting plasma glucose level was higher at baseline in patients receiving dapagliflozin and was significantly reduced only in this group (P = 0.03 for time by treatment interaction). The serum magnesium level increased after 4-week administration of dapagliflozin, whereas it was unmodified after HCT treatment (Table 1). Conversely, the remaining electrolyte levels, lipid profile, and estimated glomerular filtration rate were comparable at baseline in the two groups and were not affected by either treatment. Dapagliflozin induced a significant rise in plasma aldosterone levels; plasma renin activity and adrenaline and noradrenaline levels did not change after either treatment (Table 2). Table 2. Anthropometric and Biochemical Variables Before and After 4-Wk Treatment With Either Dapagliflozin or HCT Dapagliflozin (n = 20) HCT (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Age, y 60 ± 8 NA 62 ± 8 NA Sex, M/F 12/8 NA 14/6 NA BMI, kg/m2 32.38 ± 6.72 31.69 ± 6.81 28.79 ± 4.88 28.98 ± 4.48 0.149 0.563 0.074 Office systolic BP, mmHg 135.9 ± 11.4 134.1 ± 14.6 139.3 ± 15.9 134.6 ± 16.5 0.748 0.177 0.773 Office diastolic BP, mmHg 75.6 ± 6.5 73.1 ± 8.0 76.2 ± 10.1 73.9 ± 7.2 0.867 0.104 0.758 Fasting plasma glucose, mmol/L 8.16 ± 2.04b 7.34 ± 1.83 6.92 ± 1.57 7.25 ± 2.74 0.367 0.469 0.046 HbA1c, mmol/mol 58.9 ± 14.5 NA 52.3 ± 12.2 NA Hematocrit, % 41.82 ± 3.34 41.84 ± 2.92 42.39 ± 2.67 41.85 ± 3.15 0.767 0.303 0.162 Total cholesterol, mmol/L 4.37 ± 0.73 4.37 ± 0.57 4.25 ± 0.54 4.20 ± 0.74 0.378 0.641 0.963 HDL cholesterol, mmol/L 1.28 ± 0.40 1.31 ± 0.47 1.42 ± 0.43 1.32 ± 0.36 0.607 0.278 0.115 Triacylglycerol, mmol/L 1.44 ± 0.58 1.56 ± 0.76 1.15 ± 0.51 1.23 ± 0.55 0.148 0.130 0.623 eGFR, mL/min/1.73m2 94.7 ± 11.9 99.3 ± 16.9 91.2 ± 10.9 91.8 ± 12.4 0.179 0.792 0.157 Serum electrolytes Na, mmol/L 139.95 ± 2.11 139.61 ± 1.54 139.56 ± 1.93 139.88 ± 2.13 0.953 0.967 0.480 K, mmol/L 4.55 ± 0.33 4.97 ± 0.36 4.57 ± 0.35 4.47 ± 0.31 0.927 0.109 0.383 Ca, mg/dL 2.39 ± 0.10 2.39 ± 0.09 2.41 ± 0.12 2.43 ± 0.13 0.391 0.420 0.793 Cl−, mmol/L 99.95 ± 2.72 99.78 ± 2.16 99.81 ± 2.81 99.53 ± 2.63 0.922 0.703 0.702 Mg, mmol/L 0.94 ± 0.14 1.00 ± 0.11c 0.95 ± 0.14 0.95 ± 0.12 0.481 0.064 0.018 Osmolarity, mOsm/L 294.9 ± 5.0 293.6 ± 4.0 292.5 ± 4.6 294.8 ± 4.6 0.684 0.476 0.026 Plasma renin activity, ng/mL/h 2.18 ± 3.31 3.19 ± 3.56 2.72 ± 3.12 5.15 ± 6.67 0.232 0.156 0.553 Aldosterone, pg/mL 1.14 ± 0.55 1.79 ± 1.09b,c 1.44 ± 0.95 1.01 ± 0.43 0.276 0.575 0.007 Norepinephrine, pmol/L 638.2 ± 224.1 574.9 ± 275.0 755.8 ± 231.2 765.9 ± 282.6 0.072 0.533 0.380 Adrenaline, pmol/L 47.0 ± 38.8 33.4 ± 19.4 56.3 ± 38.2 50.9 ± 28.7 0.221 0.085 0.472 Dapagliflozin (n = 20) HCT (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Age, y 60 ± 8 NA 62 ± 8 NA Sex, M/F 12/8 NA 14/6 NA BMI, kg/m2 32.38 ± 6.72 31.69 ± 6.81 28.79 ± 4.88 28.98 ± 4.48 0.149 0.563 0.074 Office systolic BP, mmHg 135.9 ± 11.4 134.1 ± 14.6 139.3 ± 15.9 134.6 ± 16.5 0.748 0.177 0.773 Office diastolic BP, mmHg 75.6 ± 6.5 73.1 ± 8.0 76.2 ± 10.1 73.9 ± 7.2 0.867 0.104 0.758 Fasting plasma glucose, mmol/L 8.16 ± 2.04b 7.34 ± 1.83 6.92 ± 1.57 7.25 ± 2.74 0.367 0.469 0.046 HbA1c, mmol/mol 58.9 ± 14.5 NA 52.3 ± 12.2 NA Hematocrit, % 41.82 ± 3.34 41.84 ± 2.92 42.39 ± 2.67 41.85 ± 3.15 0.767 0.303 0.162 Total cholesterol, mmol/L 4.37 ± 0.73 4.37 ± 0.57 4.25 ± 0.54 4.20 ± 0.74 0.378 0.641 0.963 HDL cholesterol, mmol/L 1.28 ± 0.40 1.31 ± 0.47 1.42 ± 0.43 1.32 ± 0.36 0.607 0.278 0.115 Triacylglycerol, mmol/L 1.44 ± 0.58 1.56 ± 0.76 1.15 ± 0.51 1.23 ± 0.55 0.148 0.130 0.623 eGFR, mL/min/1.73m2 94.7 ± 11.9 99.3 ± 16.9 91.2 ± 10.9 91.8 ± 12.4 0.179 0.792 0.157 Serum electrolytes Na, mmol/L 139.95 ± 2.11 139.61 ± 1.54 139.56 ± 1.93 139.88 ± 2.13 0.953 0.967 0.480 K, mmol/L 4.55 ± 0.33 4.97 ± 0.36 4.57 ± 0.35 4.47 ± 0.31 0.927 0.109 0.383 Ca, mg/dL 2.39 ± 0.10 2.39 ± 0.09 2.41 ± 0.12 2.43 ± 0.13 0.391 0.420 0.793 Cl−, mmol/L 99.95 ± 2.72 99.78 ± 2.16 99.81 ± 2.81 99.53 ± 2.63 0.922 0.703 0.702 Mg, mmol/L 0.94 ± 0.14 1.00 ± 0.11c 0.95 ± 0.14 0.95 ± 0.12 0.481 0.064 0.018 Osmolarity, mOsm/L 294.9 ± 5.0 293.6 ± 4.0 292.5 ± 4.6 294.8 ± 4.6 0.684 0.476 0.026 Plasma renin activity, ng/mL/h 2.18 ± 3.31 3.19 ± 3.56 2.72 ± 3.12 5.15 ± 6.67 0.232 0.156 0.553 Aldosterone, pg/mL 1.14 ± 0.55 1.79 ± 1.09b,c 1.44 ± 0.95 1.01 ± 0.43 0.276 0.575 0.007 Norepinephrine, pmol/L 638.2 ± 224.1 574.9 ± 275.0 755.8 ± 231.2 765.9 ± 282.6 0.072 0.533 0.380 Adrenaline, pmol/L 47.0 ± 38.8 33.4 ± 19.4 56.3 ± 38.2 50.9 ± 28.7 0.221 0.085 0.472 Data reported as mean ± SD unless otherwise indicated. Abbreviations: BMI, body mass index; eGFR, estimated glomerular filtration rate; HDL, high-density lipoprotein; NA, not applicable. a P values in bold type are statistically significant at P < 0.05. b P < 0.05 for the comparison between dapagliflozin and HCT. c P < 0.05 for the comparison between visits 1 and 0. Open in new tab Table 2. Anthropometric and Biochemical Variables Before and After 4-Wk Treatment With Either Dapagliflozin or HCT Dapagliflozin (n = 20) HCT (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Age, y 60 ± 8 NA 62 ± 8 NA Sex, M/F 12/8 NA 14/6 NA BMI, kg/m2 32.38 ± 6.72 31.69 ± 6.81 28.79 ± 4.88 28.98 ± 4.48 0.149 0.563 0.074 Office systolic BP, mmHg 135.9 ± 11.4 134.1 ± 14.6 139.3 ± 15.9 134.6 ± 16.5 0.748 0.177 0.773 Office diastolic BP, mmHg 75.6 ± 6.5 73.1 ± 8.0 76.2 ± 10.1 73.9 ± 7.2 0.867 0.104 0.758 Fasting plasma glucose, mmol/L 8.16 ± 2.04b 7.34 ± 1.83 6.92 ± 1.57 7.25 ± 2.74 0.367 0.469 0.046 HbA1c, mmol/mol 58.9 ± 14.5 NA 52.3 ± 12.2 NA Hematocrit, % 41.82 ± 3.34 41.84 ± 2.92 42.39 ± 2.67 41.85 ± 3.15 0.767 0.303 0.162 Total cholesterol, mmol/L 4.37 ± 0.73 4.37 ± 0.57 4.25 ± 0.54 4.20 ± 0.74 0.378 0.641 0.963 HDL cholesterol, mmol/L 1.28 ± 0.40 1.31 ± 0.47 1.42 ± 0.43 1.32 ± 0.36 0.607 0.278 0.115 Triacylglycerol, mmol/L 1.44 ± 0.58 1.56 ± 0.76 1.15 ± 0.51 1.23 ± 0.55 0.148 0.130 0.623 eGFR, mL/min/1.73m2 94.7 ± 11.9 99.3 ± 16.9 91.2 ± 10.9 91.8 ± 12.4 0.179 0.792 0.157 Serum electrolytes Na, mmol/L 139.95 ± 2.11 139.61 ± 1.54 139.56 ± 1.93 139.88 ± 2.13 0.953 0.967 0.480 K, mmol/L 4.55 ± 0.33 4.97 ± 0.36 4.57 ± 0.35 4.47 ± 0.31 0.927 0.109 0.383 Ca, mg/dL 2.39 ± 0.10 2.39 ± 0.09 2.41 ± 0.12 2.43 ± 0.13 0.391 0.420 0.793 Cl−, mmol/L 99.95 ± 2.72 99.78 ± 2.16 99.81 ± 2.81 99.53 ± 2.63 0.922 0.703 0.702 Mg, mmol/L 0.94 ± 0.14 1.00 ± 0.11c 0.95 ± 0.14 0.95 ± 0.12 0.481 0.064 0.018 Osmolarity, mOsm/L 294.9 ± 5.0 293.6 ± 4.0 292.5 ± 4.6 294.8 ± 4.6 0.684 0.476 0.026 Plasma renin activity, ng/mL/h 2.18 ± 3.31 3.19 ± 3.56 2.72 ± 3.12 5.15 ± 6.67 0.232 0.156 0.553 Aldosterone, pg/mL 1.14 ± 0.55 1.79 ± 1.09b,c 1.44 ± 0.95 1.01 ± 0.43 0.276 0.575 0.007 Norepinephrine, pmol/L 638.2 ± 224.1 574.9 ± 275.0 755.8 ± 231.2 765.9 ± 282.6 0.072 0.533 0.380 Adrenaline, pmol/L 47.0 ± 38.8 33.4 ± 19.4 56.3 ± 38.2 50.9 ± 28.7 0.221 0.085 0.472 Dapagliflozin (n = 20) HCT (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Age, y 60 ± 8 NA 62 ± 8 NA Sex, M/F 12/8 NA 14/6 NA BMI, kg/m2 32.38 ± 6.72 31.69 ± 6.81 28.79 ± 4.88 28.98 ± 4.48 0.149 0.563 0.074 Office systolic BP, mmHg 135.9 ± 11.4 134.1 ± 14.6 139.3 ± 15.9 134.6 ± 16.5 0.748 0.177 0.773 Office diastolic BP, mmHg 75.6 ± 6.5 73.1 ± 8.0 76.2 ± 10.1 73.9 ± 7.2 0.867 0.104 0.758 Fasting plasma glucose, mmol/L 8.16 ± 2.04b 7.34 ± 1.83 6.92 ± 1.57 7.25 ± 2.74 0.367 0.469 0.046 HbA1c, mmol/mol 58.9 ± 14.5 NA 52.3 ± 12.2 NA Hematocrit, % 41.82 ± 3.34 41.84 ± 2.92 42.39 ± 2.67 41.85 ± 3.15 0.767 0.303 0.162 Total cholesterol, mmol/L 4.37 ± 0.73 4.37 ± 0.57 4.25 ± 0.54 4.20 ± 0.74 0.378 0.641 0.963 HDL cholesterol, mmol/L 1.28 ± 0.40 1.31 ± 0.47 1.42 ± 0.43 1.32 ± 0.36 0.607 0.278 0.115 Triacylglycerol, mmol/L 1.44 ± 0.58 1.56 ± 0.76 1.15 ± 0.51 1.23 ± 0.55 0.148 0.130 0.623 eGFR, mL/min/1.73m2 94.7 ± 11.9 99.3 ± 16.9 91.2 ± 10.9 91.8 ± 12.4 0.179 0.792 0.157 Serum electrolytes Na, mmol/L 139.95 ± 2.11 139.61 ± 1.54 139.56 ± 1.93 139.88 ± 2.13 0.953 0.967 0.480 K, mmol/L 4.55 ± 0.33 4.97 ± 0.36 4.57 ± 0.35 4.47 ± 0.31 0.927 0.109 0.383 Ca, mg/dL 2.39 ± 0.10 2.39 ± 0.09 2.41 ± 0.12 2.43 ± 0.13 0.391 0.420 0.793 Cl−, mmol/L 99.95 ± 2.72 99.78 ± 2.16 99.81 ± 2.81 99.53 ± 2.63 0.922 0.703 0.702 Mg, mmol/L 0.94 ± 0.14 1.00 ± 0.11c 0.95 ± 0.14 0.95 ± 0.12 0.481 0.064 0.018 Osmolarity, mOsm/L 294.9 ± 5.0 293.6 ± 4.0 292.5 ± 4.6 294.8 ± 4.6 0.684 0.476 0.026 Plasma renin activity, ng/mL/h 2.18 ± 3.31 3.19 ± 3.56 2.72 ± 3.12 5.15 ± 6.67 0.232 0.156 0.553 Aldosterone, pg/mL 1.14 ± 0.55 1.79 ± 1.09b,c 1.44 ± 0.95 1.01 ± 0.43 0.276 0.575 0.007 Norepinephrine, pmol/L 638.2 ± 224.1 574.9 ± 275.0 755.8 ± 231.2 765.9 ± 282.6 0.072 0.533 0.380 Adrenaline, pmol/L 47.0 ± 38.8 33.4 ± 19.4 56.3 ± 38.2 50.9 ± 28.7 0.221 0.085 0.472 Data reported as mean ± SD unless otherwise indicated. Abbreviations: BMI, body mass index; eGFR, estimated glomerular filtration rate; HDL, high-density lipoprotein; NA, not applicable. a P values in bold type are statistically significant at P < 0.05. b P < 0.05 for the comparison between dapagliflozin and HCT. c P < 0.05 for the comparison between visits 1 and 0. Open in new tab Urinary parameters are reported in Table 3. As expected, SGLT2 inhibition resulted in an increase of 24-hour diuresis, glycosuria, and osmolar clearance, whereas no substantial changes were reported in either group for urinary albumin-to-creatinine ratio and electrolytes. Table 3. Urinary Parameters Before and After 4-Wk Treatment With Either Dapagliflozin or HCT Dapagliflozin (n = 20) Hydrochlorothiazide (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Diuresis, mL/24 h 1250 (1000–2100) 2250b (1900–3000) 1750 (1250–2100) 1950 (1288–2138) 0.993 <0.001 <0.001 Albumin-to-creatinine ratio, mg/g 1.20 (0–14.75) 1.30 (0–10.68) 13.95 (0.78–20.25) 8.75 (3.98–21.93) 0.281 0.734 0.932 Urinary electrolytes Na, mEq/24 h 179 (118–235) 185 (157–213) 148 (95–229) 188 (140–240) 0.725 0.074 0.450 K, mEq/24 h 66 (46–90) 78 (56–97) 65 (45–100) 93 (64–104) 0.323 0.043 0.163 Ca, mg/24 h 222 (59–332) 236 (167–405) 194 (89–253) 185 (84–231) 0.085 0.694 0.555 Cl−, mEq/24 h 186 (122–239) 175 (159–198) 157 (93–228) 197 (130–247) 0.653 0.101 0.262 Mg, mg/24 h 70 (41–129) 110 (92–137) 77 (53–115) 88 (51–143) 0.416 0.082 0.203 Osmolarity, mOsm/L 569 ± 154 614 ± 118 479 ± 154 520 ± 154 0.057 0.114 0.945 Glycosuria, mg/24 h 180 (111–3826) 79,326b,c (59,959–147,923) 115 (67–1536) 127 (66–198) <0.001 <0.001 <0.001 Osmolar clearance, mL/24 h 2618 (2230–3275) 4324b,c (3404–5693) 2699 (1880–3587) 2995 (2537–3814) 0.061 <0.001 <0.001 Free water clearance, mL/24 h −935 (–1706 to 1099) −30 (–87 to –11) −757 (–1688 to –380) −22 (–27 to –6) 0.910 0.186 0.42 Na fractional excretion 0.77 ± 0.37 0.79 ± 0.33 0.66 ± 0.26 0.72 ± 0.26 0.503 0.241 0.613 Dapagliflozin (n = 20) Hydrochlorothiazide (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Diuresis, mL/24 h 1250 (1000–2100) 2250b (1900–3000) 1750 (1250–2100) 1950 (1288–2138) 0.993 <0.001 <0.001 Albumin-to-creatinine ratio, mg/g 1.20 (0–14.75) 1.30 (0–10.68) 13.95 (0.78–20.25) 8.75 (3.98–21.93) 0.281 0.734 0.932 Urinary electrolytes Na, mEq/24 h 179 (118–235) 185 (157–213) 148 (95–229) 188 (140–240) 0.725 0.074 0.450 K, mEq/24 h 66 (46–90) 78 (56–97) 65 (45–100) 93 (64–104) 0.323 0.043 0.163 Ca, mg/24 h 222 (59–332) 236 (167–405) 194 (89–253) 185 (84–231) 0.085 0.694 0.555 Cl−, mEq/24 h 186 (122–239) 175 (159–198) 157 (93–228) 197 (130–247) 0.653 0.101 0.262 Mg, mg/24 h 70 (41–129) 110 (92–137) 77 (53–115) 88 (51–143) 0.416 0.082 0.203 Osmolarity, mOsm/L 569 ± 154 614 ± 118 479 ± 154 520 ± 154 0.057 0.114 0.945 Glycosuria, mg/24 h 180 (111–3826) 79,326b,c (59,959–147,923) 115 (67–1536) 127 (66–198) <0.001 <0.001 <0.001 Osmolar clearance, mL/24 h 2618 (2230–3275) 4324b,c (3404–5693) 2699 (1880–3587) 2995 (2537–3814) 0.061 <0.001 <0.001 Free water clearance, mL/24 h −935 (–1706 to 1099) −30 (–87 to –11) −757 (–1688 to –380) −22 (–27 to –6) 0.910 0.186 0.42 Na fractional excretion 0.77 ± 0.37 0.79 ± 0.33 0.66 ± 0.26 0.72 ± 0.26 0.503 0.241 0.613 Data are reported as mean ± SD or median (interquartile range). a P values in bold type are statistically significant at P < 0.05. b P < 0.05 for the comparison between visits 1 and 0. c P < 0.05 for the comparison between dapagliflozin and HCT. Open in new tab Table 3. Urinary Parameters Before and After 4-Wk Treatment With Either Dapagliflozin or HCT Dapagliflozin (n = 20) Hydrochlorothiazide (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Diuresis, mL/24 h 1250 (1000–2100) 2250b (1900–3000) 1750 (1250–2100) 1950 (1288–2138) 0.993 <0.001 <0.001 Albumin-to-creatinine ratio, mg/g 1.20 (0–14.75) 1.30 (0–10.68) 13.95 (0.78–20.25) 8.75 (3.98–21.93) 0.281 0.734 0.932 Urinary electrolytes Na, mEq/24 h 179 (118–235) 185 (157–213) 148 (95–229) 188 (140–240) 0.725 0.074 0.450 K, mEq/24 h 66 (46–90) 78 (56–97) 65 (45–100) 93 (64–104) 0.323 0.043 0.163 Ca, mg/24 h 222 (59–332) 236 (167–405) 194 (89–253) 185 (84–231) 0.085 0.694 0.555 Cl−, mEq/24 h 186 (122–239) 175 (159–198) 157 (93–228) 197 (130–247) 0.653 0.101 0.262 Mg, mg/24 h 70 (41–129) 110 (92–137) 77 (53–115) 88 (51–143) 0.416 0.082 0.203 Osmolarity, mOsm/L 569 ± 154 614 ± 118 479 ± 154 520 ± 154 0.057 0.114 0.945 Glycosuria, mg/24 h 180 (111–3826) 79,326b,c (59,959–147,923) 115 (67–1536) 127 (66–198) <0.001 <0.001 <0.001 Osmolar clearance, mL/24 h 2618 (2230–3275) 4324b,c (3404–5693) 2699 (1880–3587) 2995 (2537–3814) 0.061 <0.001 <0.001 Free water clearance, mL/24 h −935 (–1706 to 1099) −30 (–87 to –11) −757 (–1688 to –380) −22 (–27 to –6) 0.910 0.186 0.42 Na fractional excretion 0.77 ± 0.37 0.79 ± 0.33 0.66 ± 0.26 0.72 ± 0.26 0.503 0.241 0.613 Dapagliflozin (n = 20) Hydrochlorothiazide (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Diuresis, mL/24 h 1250 (1000–2100) 2250b (1900–3000) 1750 (1250–2100) 1950 (1288–2138) 0.993 <0.001 <0.001 Albumin-to-creatinine ratio, mg/g 1.20 (0–14.75) 1.30 (0–10.68) 13.95 (0.78–20.25) 8.75 (3.98–21.93) 0.281 0.734 0.932 Urinary electrolytes Na, mEq/24 h 179 (118–235) 185 (157–213) 148 (95–229) 188 (140–240) 0.725 0.074 0.450 K, mEq/24 h 66 (46–90) 78 (56–97) 65 (45–100) 93 (64–104) 0.323 0.043 0.163 Ca, mg/24 h 222 (59–332) 236 (167–405) 194 (89–253) 185 (84–231) 0.085 0.694 0.555 Cl−, mEq/24 h 186 (122–239) 175 (159–198) 157 (93–228) 197 (130–247) 0.653 0.101 0.262 Mg, mg/24 h 70 (41–129) 110 (92–137) 77 (53–115) 88 (51–143) 0.416 0.082 0.203 Osmolarity, mOsm/L 569 ± 154 614 ± 118 479 ± 154 520 ± 154 0.057 0.114 0.945 Glycosuria, mg/24 h 180 (111–3826) 79,326b,c (59,959–147,923) 115 (67–1536) 127 (66–198) <0.001 <0.001 <0.001 Osmolar clearance, mL/24 h 2618 (2230–3275) 4324b,c (3404–5693) 2699 (1880–3587) 2995 (2537–3814) 0.061 <0.001 <0.001 Free water clearance, mL/24 h −935 (–1706 to 1099) −30 (–87 to –11) −757 (–1688 to –380) −22 (–27 to –6) 0.910 0.186 0.42 Na fractional excretion 0.77 ± 0.37 0.79 ± 0.33 0.66 ± 0.26 0.72 ± 0.26 0.503 0.241 0.613 Data are reported as mean ± SD or median (interquartile range). a P values in bold type are statistically significant at P < 0.05. b P < 0.05 for the comparison between visits 1 and 0. c P < 0.05 for the comparison between dapagliflozin and HCT. Open in new tab Data from noninvasive vascular assessment are reported in Table 4. Endothelium-dependent and -independent vasodilation, as explored by FMD and response to nitrates, respectively, did not change after either treatment. Large artery stiffness (carotid-femoral PWV) was also unaffected in both groups. With respect to renal vascular parameters, the RI did not show any variation after treatments; interestingly, the DRIN (i.e., the relative change after nitrate administration) remained unmodified in the dapagliflozin group, whereas it tended to increase in the HCT group (P = 0.047 for time by group interaction; Fig. 1). Central BP tended to be reduced after dapagliflozin and HCT treatment without inducing a change in heart rate. Table 4. Vascular Parameters Before and After 4-Wk Treatment With Either Dapagliflozin or HCT Dapagliflozin (n = 20) HCT (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Baseline brachial artery diameter, FMD, mm 4.18 ± 0.63 4.18 ± 0.54 4.52 ± 0.61 4.56 ± 0.63 0.088 0.869 0.653 Maximum brachial artery diameter, FMD, mm 4.43 ± 0.64 4.32 ± 0.58 4.70 ± 0.58 4.60 ± 0.53 0.297 0.169 0.252 Baseline shear stress per secondb 268.6 ± 110.4 220.8 ± 57.2 238.5 ± 108.3 208.3 ± 73.1 0.451 0.080 0.932 Hyperemic shear stress per second 925.0 ± 380.3 805.5 ± 259.5 839.2 ± 354.0 779.9 ± 274.8 0.744 0.277 0.568 FMD, % 3.80 ± 1.87 2.98 ± 1.91 3.27 ± 2.00 2.88 ± 2.05 0.643 0.210 0.425 Baseline brachial artery diameter, GTN, mm 4.32 ± 0.57 4.17 ± 0.51 4.47 ± 0.46 4.45 ± 0.58 0.364 0.231 0.331 Maximum brachial artery diameter, GTN, mm 4.59 ± 0.60 4.46 ± 0.55 4.76 ± 0.54 4.73 ± 0.55 0.353 0.262 0.273 Response to GTN, % 6.08 ± 2.69 6.73 ± 3.87 5.89 ± 3.87 5.96 ± 4.46 0.689 0.631 0.967 Carotid-femoral PWV, m/s × 0.8 10.35 ± 1.64 10.18 ± 1.47 11.15 ± 2.61 10.63 ± 2.65 0.387 0.202 0.640 AIx, % 27.90 ± 8.14 23.67 ± 10.11 26.44 ± 11.93 26.31 ± 9.05 0.863 0.130 0.147 AIx@75,c % 24.15 ± 7.28 20.89 ± 9.49 24.88 ± 11.50 23.44 ± 7.97 0.599 0.099 0.477 Aortic systolic BP, mm Hg 123.1 ± 9.6 119.9 ± 13.7 128.7 ± 17.3 122.1 ± 15.7 0.399 0.062 0.672 Aortic diastolic BP, mm Hg 79.2 ± 11.4 74.5 ± 8.4 79.6 ± 13.7 75.4 ± 7.6 0.958 0.051 0.767 Aortic PP, mm Hg 43.90 ± 11.96 45.39 ± 9.94 49.06 ± 14.89 46.69 ± 13.54 0.395 0.849 0.402 Heart rate, bpm 67.15 ± 8.62 69.11 ± 9.62 71.31 ± 9.98 69.00 ± 8.45 0.544 0.745 0.139 Renal RI 0.65 ± 0.05 0.64 ± 0.06 0.64 ± 0.04 0.62 ± 0.05 0.657 0.002 0.265 DRIN, % −3.36 ± 3.19 −3.70 ± 3.49 −5.60 ± 4.43 −3.36 ± 3.88 0.308 0.495 0.047 Dapagliflozin (n = 20) HCT (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Baseline brachial artery diameter, FMD, mm 4.18 ± 0.63 4.18 ± 0.54 4.52 ± 0.61 4.56 ± 0.63 0.088 0.869 0.653 Maximum brachial artery diameter, FMD, mm 4.43 ± 0.64 4.32 ± 0.58 4.70 ± 0.58 4.60 ± 0.53 0.297 0.169 0.252 Baseline shear stress per secondb 268.6 ± 110.4 220.8 ± 57.2 238.5 ± 108.3 208.3 ± 73.1 0.451 0.080 0.932 Hyperemic shear stress per second 925.0 ± 380.3 805.5 ± 259.5 839.2 ± 354.0 779.9 ± 274.8 0.744 0.277 0.568 FMD, % 3.80 ± 1.87 2.98 ± 1.91 3.27 ± 2.00 2.88 ± 2.05 0.643 0.210 0.425 Baseline brachial artery diameter, GTN, mm 4.32 ± 0.57 4.17 ± 0.51 4.47 ± 0.46 4.45 ± 0.58 0.364 0.231 0.331 Maximum brachial artery diameter, GTN, mm 4.59 ± 0.60 4.46 ± 0.55 4.76 ± 0.54 4.73 ± 0.55 0.353 0.262 0.273 Response to GTN, % 6.08 ± 2.69 6.73 ± 3.87 5.89 ± 3.87 5.96 ± 4.46 0.689 0.631 0.967 Carotid-femoral PWV, m/s × 0.8 10.35 ± 1.64 10.18 ± 1.47 11.15 ± 2.61 10.63 ± 2.65 0.387 0.202 0.640 AIx, % 27.90 ± 8.14 23.67 ± 10.11 26.44 ± 11.93 26.31 ± 9.05 0.863 0.130 0.147 AIx@75,c % 24.15 ± 7.28 20.89 ± 9.49 24.88 ± 11.50 23.44 ± 7.97 0.599 0.099 0.477 Aortic systolic BP, mm Hg 123.1 ± 9.6 119.9 ± 13.7 128.7 ± 17.3 122.1 ± 15.7 0.399 0.062 0.672 Aortic diastolic BP, mm Hg 79.2 ± 11.4 74.5 ± 8.4 79.6 ± 13.7 75.4 ± 7.6 0.958 0.051 0.767 Aortic PP, mm Hg 43.90 ± 11.96 45.39 ± 9.94 49.06 ± 14.89 46.69 ± 13.54 0.395 0.849 0.402 Heart rate, bpm 67.15 ± 8.62 69.11 ± 9.62 71.31 ± 9.98 69.00 ± 8.45 0.544 0.745 0.139 Renal RI 0.65 ± 0.05 0.64 ± 0.06 0.64 ± 0.04 0.62 ± 0.05 0.657 0.002 0.265 DRIN, % −3.36 ± 3.19 −3.70 ± 3.49 −5.60 ± 4.43 −3.36 ± 3.88 0.308 0.495 0.047 Data in the dapagliflozin and HCT columns are reported as mean ± SD. Abbreviation: PP, pulse pressure. a P values in bold type are statistically significant at P < 0.05. b Shear stress is the frictional force at the endothelial surface produced by flowing blood, directly related to low and blood viscosity. c Alx@75 is the heart rate-corrected augmentation index. Open in new tab Table 4. Vascular Parameters Before and After 4-Wk Treatment With Either Dapagliflozin or HCT Dapagliflozin (n = 20) HCT (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Baseline brachial artery diameter, FMD, mm 4.18 ± 0.63 4.18 ± 0.54 4.52 ± 0.61 4.56 ± 0.63 0.088 0.869 0.653 Maximum brachial artery diameter, FMD, mm 4.43 ± 0.64 4.32 ± 0.58 4.70 ± 0.58 4.60 ± 0.53 0.297 0.169 0.252 Baseline shear stress per secondb 268.6 ± 110.4 220.8 ± 57.2 238.5 ± 108.3 208.3 ± 73.1 0.451 0.080 0.932 Hyperemic shear stress per second 925.0 ± 380.3 805.5 ± 259.5 839.2 ± 354.0 779.9 ± 274.8 0.744 0.277 0.568 FMD, % 3.80 ± 1.87 2.98 ± 1.91 3.27 ± 2.00 2.88 ± 2.05 0.643 0.210 0.425 Baseline brachial artery diameter, GTN, mm 4.32 ± 0.57 4.17 ± 0.51 4.47 ± 0.46 4.45 ± 0.58 0.364 0.231 0.331 Maximum brachial artery diameter, GTN, mm 4.59 ± 0.60 4.46 ± 0.55 4.76 ± 0.54 4.73 ± 0.55 0.353 0.262 0.273 Response to GTN, % 6.08 ± 2.69 6.73 ± 3.87 5.89 ± 3.87 5.96 ± 4.46 0.689 0.631 0.967 Carotid-femoral PWV, m/s × 0.8 10.35 ± 1.64 10.18 ± 1.47 11.15 ± 2.61 10.63 ± 2.65 0.387 0.202 0.640 AIx, % 27.90 ± 8.14 23.67 ± 10.11 26.44 ± 11.93 26.31 ± 9.05 0.863 0.130 0.147 AIx@75,c % 24.15 ± 7.28 20.89 ± 9.49 24.88 ± 11.50 23.44 ± 7.97 0.599 0.099 0.477 Aortic systolic BP, mm Hg 123.1 ± 9.6 119.9 ± 13.7 128.7 ± 17.3 122.1 ± 15.7 0.399 0.062 0.672 Aortic diastolic BP, mm Hg 79.2 ± 11.4 74.5 ± 8.4 79.6 ± 13.7 75.4 ± 7.6 0.958 0.051 0.767 Aortic PP, mm Hg 43.90 ± 11.96 45.39 ± 9.94 49.06 ± 14.89 46.69 ± 13.54 0.395 0.849 0.402 Heart rate, bpm 67.15 ± 8.62 69.11 ± 9.62 71.31 ± 9.98 69.00 ± 8.45 0.544 0.745 0.139 Renal RI 0.65 ± 0.05 0.64 ± 0.06 0.64 ± 0.04 0.62 ± 0.05 0.657 0.002 0.265 DRIN, % −3.36 ± 3.19 −3.70 ± 3.49 −5.60 ± 4.43 −3.36 ± 3.88 0.308 0.495 0.047 Dapagliflozin (n = 20) HCT (n = 20) P Valuea Visit 0 Visit 1 Visit 0 Visit 1 Group Time Time × Group Baseline brachial artery diameter, FMD, mm 4.18 ± 0.63 4.18 ± 0.54 4.52 ± 0.61 4.56 ± 0.63 0.088 0.869 0.653 Maximum brachial artery diameter, FMD, mm 4.43 ± 0.64 4.32 ± 0.58 4.70 ± 0.58 4.60 ± 0.53 0.297 0.169 0.252 Baseline shear stress per secondb 268.6 ± 110.4 220.8 ± 57.2 238.5 ± 108.3 208.3 ± 73.1 0.451 0.080 0.932 Hyperemic shear stress per second 925.0 ± 380.3 805.5 ± 259.5 839.2 ± 354.0 779.9 ± 274.8 0.744 0.277 0.568 FMD, % 3.80 ± 1.87 2.98 ± 1.91 3.27 ± 2.00 2.88 ± 2.05 0.643 0.210 0.425 Baseline brachial artery diameter, GTN, mm 4.32 ± 0.57 4.17 ± 0.51 4.47 ± 0.46 4.45 ± 0.58 0.364 0.231 0.331 Maximum brachial artery diameter, GTN, mm 4.59 ± 0.60 4.46 ± 0.55 4.76 ± 0.54 4.73 ± 0.55 0.353 0.262 0.273 Response to GTN, % 6.08 ± 2.69 6.73 ± 3.87 5.89 ± 3.87 5.96 ± 4.46 0.689 0.631 0.967 Carotid-femoral PWV, m/s × 0.8 10.35 ± 1.64 10.18 ± 1.47 11.15 ± 2.61 10.63 ± 2.65 0.387 0.202 0.640 AIx, % 27.90 ± 8.14 23.67 ± 10.11 26.44 ± 11.93 26.31 ± 9.05 0.863 0.130 0.147 AIx@75,c % 24.15 ± 7.28 20.89 ± 9.49 24.88 ± 11.50 23.44 ± 7.97 0.599 0.099 0.477 Aortic systolic BP, mm Hg 123.1 ± 9.6 119.9 ± 13.7 128.7 ± 17.3 122.1 ± 15.7 0.399 0.062 0.672 Aortic diastolic BP, mm Hg 79.2 ± 11.4 74.5 ± 8.4 79.6 ± 13.7 75.4 ± 7.6 0.958 0.051 0.767 Aortic PP, mm Hg 43.90 ± 11.96 45.39 ± 9.94 49.06 ± 14.89 46.69 ± 13.54 0.395 0.849 0.402 Heart rate, bpm 67.15 ± 8.62 69.11 ± 9.62 71.31 ± 9.98 69.00 ± 8.45 0.544 0.745 0.139 Renal RI 0.65 ± 0.05 0.64 ± 0.06 0.64 ± 0.04 0.62 ± 0.05 0.657 0.002 0.265 DRIN, % −3.36 ± 3.19 −3.70 ± 3.49 −5.60 ± 4.43 −3.36 ± 3.88 0.308 0.495 0.047 Data in the dapagliflozin and HCT columns are reported as mean ± SD. Abbreviation: PP, pulse pressure. a P values in bold type are statistically significant at P < 0.05. b Shear stress is the frictional force at the endothelial surface produced by flowing blood, directly related to low and blood viscosity. c Alx@75 is the heart rate-corrected augmentation index. Open in new tab Figure 1. Open in new tabDownload slide DRIN at baseline and after treatment with dapagliflozin or HCT. Figure 1. Open in new tabDownload slide DRIN at baseline and after treatment with dapagliflozin or HCT. No correlation between changes in vascular parameters and baseline levels of Na and volume status, RAS, neurohormones, and BP values was found (data not shown); no correlations with their changes with treatment was found either. Finally, we tested whether miRs potentially involved in cardiorenal function could be modified by dapagliflozin and whether they could be used to track the improvement induced by dapagliflozin in renal arterial compliance. No significant differences between groups were found in the plasma levels of all the measured miRs at baseline (Table 5). Both treatments induced a significant increase in miR-200b, miR-27b, miR-130b-3p, and miR-27a-3p, although with a similar behavior (no significant time by treatment interaction). miR-30e-5p and miR-199a-3p, instead, were differently modulated by dapagliflozin and HCT, with a significant upregulation of the former and downregulation of the latter induced by SGLT2 inhibition (Table 4). Table 5. Baseline and Posttreatment Expression of Circulating miRs in Patients Receiving Dapagliflozin and HCT Dapagliflozin HCT V0 V1 V0 V1 P Value (Time × Group)a miR-21-5p 1.596 (2.088) 1.772 (1.395) 1.443 (1.835) 1.292 (3.206) 0.611 miR-200b 0.379 (0.580) 2.802 (4.179)b 0.375 (0.453) 2.105 (2.318)b 0.629 miR-30e-5p 1.195 (3.134) 4.004 (3.563)b 1.405 (2.062) 2.615 (1.867)c 0.012 miR-199a-3p 0.353 (0.480) 0.171 (0.181)b 0.293 (0.314) 0.418 (0.725)c 0.017 miR-27b 0.072 (0.050) 0.172 (0.209)b 0.063 (0.050) 0.184 (0.305)b 0.343 miR-130b-3p 0.074 (0.083) 0.155 (0.200)b 0.044 (0.046) 0.096 (0.190)b 0.800 miR-27a-3p 0.753 (1.105) 2.039 (2.302)b 0.615 (0.619) 2.606 (3.132)b 0.297 Dapagliflozin HCT V0 V1 V0 V1 P Value (Time × Group)a miR-21-5p 1.596 (2.088) 1.772 (1.395) 1.443 (1.835) 1.292 (3.206) 0.611 miR-200b 0.379 (0.580) 2.802 (4.179)b 0.375 (0.453) 2.105 (2.318)b 0.629 miR-30e-5p 1.195 (3.134) 4.004 (3.563)b 1.405 (2.062) 2.615 (1.867)c 0.012 miR-199a-3p 0.353 (0.480) 0.171 (0.181)b 0.293 (0.314) 0.418 (0.725)c 0.017 miR-27b 0.072 (0.050) 0.172 (0.209)b 0.063 (0.050) 0.184 (0.305)b 0.343 miR-130b-3p 0.074 (0.083) 0.155 (0.200)b 0.044 (0.046) 0.096 (0.190)b 0.800 miR-27a-3p 0.753 (1.105) 2.039 (2.302)b 0.615 (0.619) 2.606 (3.132)b 0.297 Data are reported as mean (SD) of relative expression. Abbreviations: V0, baseline; V1, posttreatment. aP values in bold type are statistically significant at P < 0.05. b P < 0.05 for the comparison between visits 1 and 0. c P < 0.05 for the comparison between dapagliflozin and HCT. Open in new tab Table 5. Baseline and Posttreatment Expression of Circulating miRs in Patients Receiving Dapagliflozin and HCT Dapagliflozin HCT V0 V1 V0 V1 P Value (Time × Group)a miR-21-5p 1.596 (2.088) 1.772 (1.395) 1.443 (1.835) 1.292 (3.206) 0.611 miR-200b 0.379 (0.580) 2.802 (4.179)b 0.375 (0.453) 2.105 (2.318)b 0.629 miR-30e-5p 1.195 (3.134) 4.004 (3.563)b 1.405 (2.062) 2.615 (1.867)c 0.012 miR-199a-3p 0.353 (0.480) 0.171 (0.181)b 0.293 (0.314) 0.418 (0.725)c 0.017 miR-27b 0.072 (0.050) 0.172 (0.209)b 0.063 (0.050) 0.184 (0.305)b 0.343 miR-130b-3p 0.074 (0.083) 0.155 (0.200)b 0.044 (0.046) 0.096 (0.190)b 0.800 miR-27a-3p 0.753 (1.105) 2.039 (2.302)b 0.615 (0.619) 2.606 (3.132)b 0.297 Dapagliflozin HCT V0 V1 V0 V1 P Value (Time × Group)a miR-21-5p 1.596 (2.088) 1.772 (1.395) 1.443 (1.835) 1.292 (3.206) 0.611 miR-200b 0.379 (0.580) 2.802 (4.179)b 0.375 (0.453) 2.105 (2.318)b 0.629 miR-30e-5p 1.195 (3.134) 4.004 (3.563)b 1.405 (2.062) 2.615 (1.867)c 0.012 miR-199a-3p 0.353 (0.480) 0.171 (0.181)b 0.293 (0.314) 0.418 (0.725)c 0.017 miR-27b 0.072 (0.050) 0.172 (0.209)b 0.063 (0.050) 0.184 (0.305)b 0.343 miR-130b-3p 0.074 (0.083) 0.155 (0.200)b 0.044 (0.046) 0.096 (0.190)b 0.800 miR-27a-3p 0.753 (1.105) 2.039 (2.302)b 0.615 (0.619) 2.606 (3.132)b 0.297 Data are reported as mean (SD) of relative expression. Abbreviations: V0, baseline; V1, posttreatment. aP values in bold type are statistically significant at P < 0.05. b P < 0.05 for the comparison between visits 1 and 0. c P < 0.05 for the comparison between dapagliflozin and HCT. Open in new tab Linear correlations emerged between the change in DRIN (DRIN posttreatment minus DRIN pretreatment) and the basal expression pattern of miR-200b and miR-27b (r = 0.43, P = 0.05; and r=0.50, P = 0.03, respectively), previously described as associated with heart failure and endothelial dysfunction, and renal damage. Such a relationship held true only considering patients receiving dapagliflozin (Fig. 2). Variation of miR-27b after treatment was inversely related to change in FMD (r = −0.474; P = 0.026) and to change in AIx (r = −0.476; P = 0.022). Figure 2, Open in new tabDownload slide Direct correlation between circulating miR-20b and miR-27b (expressed as the ln) at baseline and change in DRIN (expressed as percent variation vs baseline). Δ, change in; ln, natural logarithm. Figure 2, Open in new tabDownload slide Direct correlation between circulating miR-20b and miR-27b (expressed as the ln) at baseline and change in DRIN (expressed as percent variation vs baseline). Δ, change in; ln, natural logarithm. Discussion Dapagliflozin is a widely used antihyperglycemic drug with peculiar and partially unexplained CV and renal protective effects. The main results of this study suggest two novel mechanisms of cardiorenal protection associated with dapagliflozin treatment. First, dapagliflozin upregulates the expression of miR30e-5p and miR199a-3p, which are involved in the pathophysiology of heart failure. Second, a 4-week treatment preserved renal vasodilating capacity, an effect influenced by miRs expression pattern at baseline. These effects are independent of the BP-lowering and diuretic effects of dapagliflozin (10, 24), because they are not present in the HCT arm. In this study, we also performed an extensive evaluation of the response of the systemic and renal vasculature to dapagliflozin. Brachial artery endothelial function, acutely improved by dapagliflozin (10), was not modified by such short-term treatment. Our results somewhat resonate with those of the DEFENCE (Dapagliflozin Effectiveness on the Vascular Endothelial Function and Glycemic Control) trial (8), which showed that compared with a nonhemodynamically active comparator (metformin), dapagliflozin induced a small improvement in endothelial function after 16 weeks of treatment, but such improvement was statistically significant only in the subset of patients (n = 13) with the worse metabolic control at baseline. We might also speculate that, over a subacute observation like the present one, some contraregulatory mechanisms (e.g., via aldosterone activation) might have toned down the improvement of FMD, an index of endothelial function. It is conceivable, therefore, that amelioration of this functional parameter, as well as of large arterial stiffness (indicated by PWV), requires much longer treatment duration; thus, our results do not exclude that a direct effect on vascular function or structure is a mechanism underlying SGLT2-inhibitor–induced CV protection (1, 2). On the contrary, renal vasculature seems to be favorably modified, with a dynamic renal RI, a proxy for renal vasodilating capacity, preserved by dapagliflozin, whereas it was increased in patients treated with HCT. It is important to note that an increase in DRIN (usually a negative value) indicates an impaired renal vasodilating capacity to nitrates. Interestingly, this effect was not present after 48 hours of treatment with dapagliflozin, when acute hemodynamic effects of this drug are probably predominant (10), and might represent a long-standing effect. We have previously demonstrated that DRIN may predict albuminuria onset in individuals with hypertension and diabetes (25). To our knowledge, no studies have evaluated the effect of short-term SGLT2 inhibition on resting or dynamic renal RI. Our data add a small piece of knowledge to the panel of nephroprotective effects exerted by these drugs, reinforcing the concept of the kidney as a whole, not only the tubule, as a target of several complex physiological responses triggered by SGLT2 inhibition. miRs are small noncoding RNAs able to posttranscriptionally regulate gene expression. They are stably expressed in serum or plasma, and it has been proposed that their unique expression patterns serve as disease fingerprints in many clinical settings, including diabetes (26). Our work explored whether epigenetic markers (assessed as a panel of miRs potentially involved in CV and renal disease) might predict the vascular effects of such compounds, and whether SGLT2 inhibition might exert protective effects also via an epigenetic modulation. Interestingly, the behavior of dynamic renal RI exerted by dapagliflozin was influenced by miR-27b expression; specifically, renal vasodilating capacity was preserved under dapagliflozin treatment only when miR-27b expression was low. Previous studies demonstrated that miR-27b expression clusters with metabolic syndrome and T2D (27), and it was recently identified as marker for new-onset or progression of retinopathy in patients with type 1 diabetes (28). Furthermore, we show as basal expression level of miR-200b predicts change in DRIN and that the expression level increases after both treatments. These findings resonate with observations in the eye, where miR-200b upregulation could promote retinal endothelial cell growth and proliferation by modulating TGFβ1and VEGF expression (29). However, we cannot completely exclude that the significant correlation between changes in vascular parameters and in miRs is by chance, because of the high number of comparisons performed. Another intriguing observation was the comparison of the effect of the two treatments on a panel of miRs that potentially mediate mechanisms of heart and vascular damage. The difference we observed in the expression of miRs related to CV disease and renal impairment in patients with heart failure (30, 31) deserves attention aimed at better defining their potential role in modulating the cardioprotection exerted by SGLT2 inhibitors. In detail, miR-30e-5p, which is selectively up-regulated by dapagliflozin, inhibits myocardiocyte autophagy through modulation the ACE2 pathway in an experimental model of heart failure (32), is differentially expressed between failing and nonfailing hearts, and its expression increases in patients with heart failure responding to cardiac resynchronization therapy (33, 34). The reduced expression of miR-199a-3p induced by dapagliflozin might be also intriguing in mechanistic terms, if confirmed by other studies, because antagonizing miR-199-3p could de-repress cardiac PPARδ levels, thus restoring mitochondrial fatty acid oxidation and improving cardiac function in the failing heart (35). We would point out that such a signature is quite specific: miRs modulating epithelial-mesenchymal transition (36) or associated with renal damage in chronic (19) or acute kidney disease (37) did not mark the clinical effects of dapagliflozin, and neither were differently influenced by the treatments. This might suggest that the protective effect of dapagliflozin on renal vasodilating capacity is probably a consequence of its effect on renal microcirculation, because it is influenced by miRs whose actions have such a vascular target. Taken together, though limited by the small sample size, our observations suggest an involvement of these miRs in the vascular and renal responses to these antidiabetic drugs. It is important to note that the renal protective effect of dapagliflozin is independent of BP, because it did not occur in the HCT treatment arm. We cannot completely exclude that normalization of hyperglycemia (24) is responsible for this effect, though no correlation between changes in DRIN and blood fasting glucose level was found in this study, and in larger studies no cross-sectional association between glucose levels and DRIN was found (38). However, studies comparing the renal vascular effect of SGLT2 inhibitors with those of other antidiabetic drugs are warranted to completely exclude this hypothesis. Our study also demonstrated that dapagliflozin induced a significant increase in magnesium levels. We demonstrated in a previous study that this effect is detectable after 48 hours of treatment (10). This confirmation over a prolonged period of treatment raises the possibility that this element might contribute to the CV protection exerted by SGLT2 inhibitors and is consistent with reports in the literature showing that hypomagnesemia is an established risk factor for CV disease, events, and death in the general population and in patients with chronic kidney disease (39–42). Moreover, serum Mg2+ concentrations are inversely associated with risk for death from heart failure and coronary heart disease (43, 44), and with coronary artery calcification in patients with end-stage renal disease (45). Finally, data on renal function and electrolyte and water balance deserve attention, though our findings mainly were negative. Interestingly, we did not observe the previously reported transient increase in serum creatinine level (1). The acute estimated glomerular filtration rate reduction that usually follows the onset of SGLT2 inhibition is compatible with a resetting of the macula densa tubuloglomerular feedback, depending at least in part upon volume depletion and resulting in vasoconstriction of the afferent glomerular arterioles. It is important to note that though diuresis was increased in the dapagliflozin arm, no dehydration occurred, as indicated by preserved serum osmolarity and hematocrit. In our study, patients were advised to drink an adequate amount of water; it is possible that such simple advice might have prevented such glomerular hemodynamics rearrangement. Our results also confirm that dapagliflozin does not unbalance natriuresis (46), and this is compatible with the recent demonstration that in T2D, the contribution of SGLT2 to Na reabsorption in the proximal tubule is in the range of 14% to 19% (47), with other sodium cotransporters located along the nephron compensating for SGLT2 inhibition. Strengths of our study are (1) the evaluation of a putative epigenetic modulation of the effects of SGLT2 inhibitors; (2) a comprehensive assessment of vascular function and structure by imaging biomarkers; (3) the evaluation of Na and water balance and of RAS and neurohormonal profiles as possible confounders; and (4) the study design, including a diuretic as a comparator. However, we should acknowledge some limitations as well. First, the short duration of the study likely explains the lack of significant variations in some parameters known to be affected by SGLT2 inhibition, such as body mass index or systolic BP. Second, the small sample size did not allow us to perform a multivariable analysis. Third, the effect of the two treatments on the epigenetic signature of circulating miRs should be regarded as a preliminary observation, requiring confirmation in larger groups of patients treated with SGLT2 inhibitors for a longer time. Fourth, we did not perform any evaluation of the cardiac function, another possible target for dapagliflozin- mediated CV prevention; however, such short duration of the treatment is unlikely to have modified it. Furthermore, given the difference in baseline DRIN between the two groups, we cannot completely exclude that the observed results are attributable to the phenomenon of regression to the mean. In conclusion, a short-term treatment with dapagliflozin preserves renal vasodilating capacity, which indicates a selective beneficial effect on renal vasculature. Such vascular response to can be predicted by miR-200b and miR-27b expression. In addition, dapagliflozin favorably influences the expression of some circulating miRs involved in the pathogenesis of heart failure, an intriguing prospective area of translational research. Acknowledgments We thank patients who participated in the study. Financial Support: This study was supported by an institutional unrestricted grant to A.S. from Astra Zeneca International. Clinical Trial Information: European Union Drug Regulating Authorities Clinical Trials No. 2015-004164-11 (registered 19 November 2015). Disclosure Summary: A.S. received research grants from AstraZeneca; lecture fees from AstraZeneca, Boehringer Ingelheim, Mundipharma, and Eli Lilly and Company; and consultancy fees from Sanofi. S.T. received lecture and consultancy fees from Boehringer Ingelheim, Pfizer, Sanofi, and Servier. L.G. is cofounder and councilor of QUIPU s.r.l., Pisa, Italy, a spin-off company on the University of Pisa and the Italian National Research Council. 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