Non-steroidal mineralocorticoid receptor antagonist finerenone ameliorates mitochondrial dysfunction via PI3K/Akt/eNOS signaling pathway in diabetic tubulopathy

Abstract

Diabetic tubulopathy (DT) is a recently recognized key pathology of diabetic kidney disease (DKD). The mitochondria-centric view of DT is emerging as a vital pathological factor in different types of metabolic diseases, such as DKD. Finerenone (FIN), a novel non-steroidal mineralocorticoid receptor antagonist, attenuates kidney inflammation and fibrosis in DKD, but the precise pathomechanisms remain unclear. The role of mineralocorticoid receptor (MR) in perturbing mitochondrial function via the PI3K/Akt/eNOS signaling pathway, including mitochondrial dynamics and mitophagy, was investigated under a diabetic state and high glucose (HG) ambiance. To elucidate how the activation of MR provokes mitochondrial dysfunction in DT, human kidney proximal tubular epithelial (HK-2) cells were exposed to HG, and then mitochondrial dynamics, mitophagy, mitochondrial ROS (mitoROS), signaling molecules PI3K, Akt, Akt phosphorylation and eNOS were probed. The above molecules or proteins were also explored in the kidneys of diabetic and FIN-treated mice. FIN treatment reduced oxidative stress, mitochondrial fragmentation, and apoptosis while restoring the mitophagy via PI3K/Akt/eNOS signaling pathway in HK-2 cells exposed to HG ambiance and tubular cells of DM mice. These findings linked MR activation to mitochondrial dysfunction via PI3K/Akt/eNOS signaling pathway in DT and highlight a pivotal but previously undiscovered role of FIN in alleviating renal tubule injury for the treatment of DKD.

Keywords: Diabetic kidney disease; Diabetic tubulopathy; Finerenone; Nonsteroidal mineralocorticoid receptor antagonist; PI3K/Akt/eNOS signaling pathway.

Graphical abstract

Fig. 1

Effect of finerenone on kidney function and pathologic alterations in HFD/STZ-induced T2DM mice's kidneys. (A) The schematic representation of the study protocol. (B) Representative image of mice and the alterations in kidney morphology. The variations in the body weight (C), blood glucose (D), serum creatinine (E), and urinary ACR (F) are shown (n = 6). (G) PAS staining provides representative histology of the renal cortex. Scale bar: 20 μm. (H) the production of mitochondrial ATP in kidney. All data are shown as means ± SEM.; *P<0.05 versus the control group, ^#P < 0.05 versus the T2DM group.

Fig. 2

Finerenone alleviates the overactivation of MR via PI3K/Akt/eNOS signaling pathway in HFD/STZ-induced T2DM mice. (A) Immunohistochemistry staining for MR showed that the MR expression and activity were significantly higher in the T2DM group compared to controls, and significantly reduced in the T2DM mice given finerenone. Scale bar: 200 μm. (B) The immunohistochemistry staining for p-AKT revealed that AKT activity was lowered in the T2DM group and recovered in the finerenone group. Scale bar: 200 μm. (C) The presence of NR3C2, PI3K, AKT, p-AKT, eNOS, and KIM-1 proteins. (D–I) Quantitative analysis of NR3C2, PI3K, AKT, p-AKT, eNOS, and KIM-1 proteins and β-actin served as a loading control (n = 6). Data are shown as means ± SEM; *P<0.05 vs. the control group, ^#P < 0.05 vs. the T2DM group.

Fig. 3

Alleviation of mitochondrial abnormal dynamics, abnormal mitophagy, and apoptosis in T2DM mice in the finerenone group. (A) Mitochondria in the proximal tubules were viewed using electron microscopy. Scale bar: 1 μm. (B) The mitochondrial pro-fission protein Drp1, mostly found in renal tubules, was increased in the T2DM group, however, finerenone treatment diminished its expression. (C–D) Representative immunoblots and quantitative analysis of the mitochondrial dynamic regulatory proteins, such as Drp1, Fis, Mfn1, Mfn2, and OPA from the control, T2DM, and finerenone groups (n = 6). (E) Quantitative analysis of the aspect ratio of mitochondria (n = 6). (F–G) Representative immunoblots and quantitative analysis of the mitophagy regulating proteins, such as LC3-II, p62, Atg5, Atg7, and Beclin-1 from the above three groups (n = 6). (H) Immunofluorescence staining of LC3-II in the renal tubules from each group. (I–K) Representative immunoblots and quantitative analysis of the apoptosis-related proteins, Bax and Cyt C from the above three groups (n = 6). β-actin served as a loading control. (K–L) Apoptosis was assessed by TUNEL staining (n = 6). The nuclei were counterstained by DAPI. Data are demonstrated as means ± SEM; *P<0.05 vs. the control group, ^#P < 0.05 vs. the T2DM group.

Fig. 4

Finerenone alleviates the overactivation of MR via PI3K/Akt/eNOS signaling pathway in HK-2 cells treated with HG. (A) HK-2 cells were immuno-stained to show the localization of NR3C2 (green) and p-AKT (red). Scale bar: 10 μm. (C–I) By immunoblotting, the presence of NR3C2 and KIM-1 were increased under HG ambience, but AKT was unaffected. Expressions of PI3K, p-AKT, and eNOS were decreased. Concurrently, the expression and activity of the PI3K/Akt/eNOS pathway and KIM-1, a marker of renal tubular injury, were markedly increased in the HK-2 cells treated with finerenone. Interestingly, the overactivation of MR was also reversed (n = 3). Data are shown as means ± SEM; *P<0.05 vs. the NG group, ^#P < 0.05 vs. the HG group.

Fig. 5

Finerenone attenuates HG-induced mitochondrial dynamics and abnormal mitophagy in HK-2 cells. (A–B) Quantitative analysis of mitochondrial dynamic regulatory proteins and representative immunoblots containing Drp1, Fis, Mfn1, Mfn2, and OPA from the NG, HG, and FIN groups (n = 3). (C) Confocal images of Drp1 (green), Mfn2 (red), and LC3-II (yellow) in HK-2 cells. The nuclei were counterstained with DAPI (blue). (D–F) Quantification of the degree of Drp1, Mfn2, and LC3-II by ImageJ (n-3). (G–H) Representative immunoblots and quantitative analysis of the mitophagy regulating proteins, such as LC3-II, p62, Atg5, Atg7, and Beclin-1 from the above three groups (n = 3). Data are shown as means ± SEM; *P<0.05 vs. the NG group, ^#P < 0.05 vs. the HG group.

Fig. 6

Effect of finerenone on dysfunctional mitochondria, mitoROS generation, and HK-2 cell apoptosis. (A) MitoTracker (red) staining evaluated the mitochondrial morphology. Scale bar: 5 μm. (B) Mitochondrial and intracellular ROS production was assessed by H₂-DCFDA (green) or MitoSOX (red) staining. Scale bar: 20 μm. (C–D) The degree of mitochondrial fragmentation was quantified, and mitochondrial oxidative stress in the NG, HG, and FIN groups was evaluated by ImageJ (n = 6). (E–F) Quantitative analysis and representative immunoblots of the apoptosis-related proteins, Bax and Cyt C (n = 3). (G–H) TUNEL staining was used to determine the apoptosis. Scale bar: 50 μm. Quantitative analysis of the TUNEL-positive tubular cells in the above groups as evaluated by ImageJ (n = 3). Data are indicated as means ± SEM; *P<0.05 vs. the CON group, ^#P < 0.05 vs. the HG group.

Fig. 7

Proposed model for the signaling pathway by which MR participates in the diabetic tubulopathy via PI3K/Akt/eNOS pathway.