Empagliflozin Protects HK-2 Cells from High Glucose-Mediated Injuries via a Mitochondrial Mechanism

Abstract

Empagliflozin is known to retard the progression of kidney disease in diabetic patients. However, the underlying mechanism is incompletely understood. High glucose induces oxidative stress in renal tubules, eventually leading to mitochondrial damage. Here, we investigated whether empagliflozin exhibits protective functions in renal tubules via a mitochondrial mechanism. We used human proximal tubular cell (PTC) line HK-2 and employed western blotting, terminal deoxynucleotidyl transferase dUTP nick end labelling assay, fluorescence staining, flow cytometry, and enzyme-linked immunosorbent assay to investigate the impact of high glucose and empagliflozin on cellular apoptosis, mitochondrial morphology, and functions including mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, and adenosine triphosphate (ATP) generation. We found that PTCs were susceptible to high glucose-induced mitochondrial fragmentation, and empagliflozin ameliorated this effect via the regulation of mitochondrial fission (FIS1 and DRP1) and fusion (MFN1 and MFN2) proteins. Empagliflozin reduced the high glucose-induced cellular apoptosis and improved mitochondrial functions by restoring mitochondrial ROS production, MMP, and ATP generation. Our results suggest that empagliflozin may protect renal PTCs from high glucose-mediated injuries through a mitochondrial mechanism. This could be one of the novel mechanisms explaining the benefits demonstrated in EMPA-REG OUTCOME trial.

Keywords: diabetic kidney disease; empagliflozin; mitochondria; proximal tubular cell.

Figure 1

Empagliflozin has no negative effects on the viability of HK-2 cells. Cell viability was measured with crystal violet assay and the absorbance was analyzed at 570 nm using a microplate reader. Data were obtained from three independent experiments and are expressed as mean ± SEM. Only high glucose (30 mM) treatment led to a decrease in cell viability. * P < 0.05 versus the normal glucose (5 mM) treatment group. Glu, glucose; Empa, empagliflozin.

Figure 2

Empagliflozin rescues high glucose-induced mitochondrial fragmentation in human PTCs. HK-2 cells were cultured in 5 mM glucose (A), 30 mM glucose (B), 30 mM glucose with 100 nM empagliflozin (C), and 30 mM glucose with 500 nM empagliflozin (D). Mitochondria were stained with Mitotracker red. Magnified images of indexed mitochondria (arrow) in each treatment group are shown in (E–H). (I) The mitochondrial fission rate was higher in high glucose condition. Empagliflozin rescued this effect. Data were obtained from three independent experiments and are expressed as mean ± SEM. * P < 0.05, ** P < 0.001.

Figure 3

Impact of empagliflozin on high glucose-induced alterations in the expression levels of mitochondrial fusion/fission proteins. (A–E) The expression of MFN1, MFN2, DRP1, and FIS1 was analyzed with western blotting and normalized to the level of β-actin. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.001.

Figure 4

Empagliflozin reduces the high glucose-induced apoptosis of HK-2 cells. (A–D) Fluorescence images show positive TUNEL staining in the four treatment groups. (E) Quantitative analysis of TUNEL assay shows that high glucose markedly induces apoptosis and empagliflozin ameliorates this effect. Data are expressed as mean ± SEM. ** P < 0.001.

Figure 5

Empagliflozin improves the mitochondrial function of high glucose-treated HK-2 cells. (A) Cellular ROS production, (B) mitochondrial ROS production, (C) MMP, and (D) ATP generation in the four treatment groups. Data were obtained from three independent experiments and are expressed as mean ± SEM. * P < 0.05, ** P < 0.001.