SGLT2 Inhibition via Empagliflozin Improves Endothelial Function and Reduces Mitochondrial Oxidative Stress: Insights From Frail Hypertensive and Diabetic Patients

Abstract

Background: Frailty is a multidimensional condition often diagnosed in older adults with hypertension and diabetes, and both these conditions are associated with endothelial dysfunction and oxidative stress. We investigated the functional role of the SGLT2 (sodium glucose cotransporter 2) inhibitor empagliflozin in frail diabetic and hypertensive older adults.

Methods: We studied the effects of empagliflozin in consecutive hypertensive and diabetic older patients with frailty presenting at the ASL (local health unit of the Italian Ministry of Health) of Avellino, Italy, from March 2021 to January 2022. Moreover, we performed in vitro experiments in human endothelial cells to measure cell viability, permeability, mitochondrial Ca²⁺, and oxidative stress.

Results: We evaluated 407 patients; 325 frail elders with diabetes successfully completed the study. We propensity-score matched 75 patients treated with empagliflozin and 75 with no empagliflozin. We observed a correlation between glycemia and Montreal Cognitive Assessment (MoCA) score and between glycemia and 5-meter gait speed (5mGS). At 3-month follow-up, we detected a significant improvement in the MoCA score and in the 5mGS in patients receiving empagliflozin compared with non-treated subjects. Mechanistically, we demonstrate that empagliflozin significantly reduces mitochondrial Ca²⁺ overload and reactive oxygen species production triggered by high glucose in human endothelial cells, attenuates cellular permeability, and improves cell viability in response to oxidative stress.

Conclusions: Taken together, our data indicate that empagliflozin reduces frailty in diabetic and hypertensive patients, most likely by decreasing the mitochondrial generation of reactive oxygen species in endothelial cells.

Keywords: aging; diabetes mellitus; frailty; hyperglycemia; mitochondria; occludin; sodium-glucose transporter 2 inhibitors.

Figure 1.

Flow chart of the study.

Figure 2.

Correlation between MoCA score and glycemia (A, r²: 0.91; p<0.001) and between 5mGS and glycemia (B, r²: 0.79; p<0.001). Differences in Montreal Cognitive Assessment (MoCA) score (C), 5-meter gait speed (5mGS, D), and frailty (E) at baseline and at 3-month follow-up in empagliflozin treated and not-treated groups (*:p<0.05; **:p<0.01; ***:p<0.001).

Figure 3.

Evaluation of mitochondrial Ca²⁺ (A-E) and mitochondrial reactive oxygen species (ROS) generation (F-G) in human umbilical vascular endothelial cells (HUVECs, passages 3–7) treated for 24 hours with the indicated concentrations of glucose (Glu) and empagliflozin (Empa). Mitochondrial Ca²⁺ (A-E) was measured in at least 30 cells per group, in triplicate independent biological replicates, in response to thapsigargin (Tg, 1 μM) or after the addition of 1.8 mM Ca²⁺. To assess the production of mitochondrial ROS, cells were stained with MitoTracker Green and mitoSOX Red (F, representative images from quadruplicate experiments; dimensional bar: 20 μm), and these assays have been quantified in the panel on the right; in the violin plot, median (solid line) and quartiles (dotted lines) are indicated; *: p<0.05 vs. 5 mM Glu + vehicle; #: p<0.05 vs. 30 mM Glu + Vehicle.

Figure 4.

Effects of empagliflozin on cell permeability assessed in human brain microvascular endothelial cells (A) and mRNA levels of Occludin (B) and Claudin-5 (C); in the violin plot, median (solid line) and quartiles (dotted lines) are indicated; *: p<0.05 vs. 5 mM Glu + vehicle; #: p<0.05 vs. 30 mM Glu + vehicle. Effects of empagliflozin (1 μM) on cell viability (D) assessed in human brain microvascular endothelial cells in response to increasing doses of H₂O₂; the solid line indicates the median, whereas the dotted lines indicate the quartiles; *: p<0.05 vs. vehicle.