Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jan 24;15(3):701-713.
doi: 10.7150/ijbs.29323. eCollection 2019.

Sirt6 Suppresses High Glucose-Induced Mitochondrial Dysfunction and Apoptosis in Podocytes through AMPK Activation

Yanqin Fan  1 , Qian Yang  1 , Yingjie Yang  1 , Zhao Gao  1 , Yiqiong Ma  1 , Lu Zhang  1 , Wei Liang  1 , Guohua Ding  1
Affiliations

Sirt6 Suppresses High Glucose-Induced Mitochondrial Dysfunction and Apoptosis in Podocytes through AMPK Activation

Yanqin Fan et al. Int J Biol Sci. .

Abstract

Previous studies have shown that mitochondrial dysfunction plays an important role in high- glucose(HG)-induced podocyte injury and thus contributes to the progression of diabetic nephropathy(DN). The histone deacetylase Sirtuin6 (Sirt6) has been revealed to have an essential role in the regulation of mitochondrial function in skeletal muscle and cardiomyocytes. However, its specific role in mitochondrial homeostasis in podocytes is undetermined. Here, we aimeds to explore the physiological function of Sirt6 in podocyte mitochondria and apoptosis under HG conditions and explore the possible mechanism. Herein, we observed that Sirt6-WT-1 colocalization was suppressed in the glomeruli of patients with DN. In addition, diabetic mice exhibited reduced Sirt6 expression and AMP kinase (AMPK) dephosphorylation accompanied by mitochondrial morphological abnormalities. In vitro, podocytes exposed to HG presented with mitochondrial morphological alterations and podocyte apoptosis accompanied by Sirt6 and p-AMPK downregulation. In addition, HG promoted a decrease in mitochondrial number and an increase in mitochondrial superoxide production as well as a decreased mitochondrial membrane potential. ROS production was also increased in HG-treated podocytes. Conversely, all these mitochondrial defects induced by HG were significantly alleviated by Sirt6 plasmid transfection. Sirt6 overexpression simultaneously alleviated HG-induced podocyte apoptosis and oxidative stress, as well as increased AMPK phosphorylation. Increased levels of H3K9ac and H3K56ac induced by HG were attenuated in podocytes transfected with Sirt6 plasmids. Therefore, these results elucidated that Sirt6 protects mitochondria of podocytes and exerts anti-apoptotic effects via activating AMPK pathway. The present findings provide key insights into the pivotal role of mitochondria regulation by SIRT6 in its protective effects on podocytes.

Keywords: Sirt6; apoptosis; diabetic nephropathy; mitochondrial dysfunction; podocytes.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Sirt6 expression in glomeruli from renal biopsies of individuals with DN. (A) Representative images of immunohistochemical staining of Sirt6 in renal cortical tissue of individuals from each group (original magnification, ×400). (B) Representative images of double immunofluorescent staining of WT1 and Sirt6 in kidney sections of individuals from each group (original magnification, ×400) and quantitation of these results (n=4). *P< 0.05 relative to control. Normal=healthy subjects, DN= patients with diabetic nephropathy.
Figure 2
Figure 2
General characteristics of mice with DN. (A) Representative micrographs of HE-stained kidney sections (row 1) (original magnification, ×400), PAS-stained kidney sections (row 2) (original magnification, ×400), and transmission electron micrographs (row 3) (original magnification, ×3 000, ×8 000) from different groups. (B) Metabolic parameters including blood glucose, body weight, kidney/body weight and ACR (albumin-to-creatinine ratios) of mice in each group (n=6). *P< 0.05 relative to control. CTL=control, DN=diabetic nephropathy.
Figure 3
Figure 3
Sirt6 expression in glomeruli from mice with DN. (A) Representative Western blots of glomerular Sirt6 expression and quantitation in the different groups (n=6). *P< 0.05 relative to control. (B) The mRNA level of glomerular Sirt6 expression in each group (n=6). *P< 0.05 relative to control. (C) Representative images of double immunofluorescent staining of glomerular WT1 and Sirt6 in the different groups (original magnification, ×400) and quantitation of these results (n=6). *P< 0.05 relative to control. CTL=control, DN=diabetic nephropathy.
Figure 4
Figure 4
Effects of HG on podocyte mitochondrial morphology and function of mice with DN. (A) Representative images of the ultrastructure of podocyte mitochondria by transmission electron microscopy in each group (original magnification, ×15 000). (B) Representative Western blots of glomerular p-AMPK and AMPK expression in the different groups and quantitation of these results (n=6). *P< 0.05 relative to control. CTL=control, DN=diabetic nephropathy.
Figure 5
Figure 5
Effects of HG on Sirt6 expression in cultured podocytes. (A) Representative Western blots of Sirt6 expression in cultured podocytes stimulated with different concentrations of glucose for 24 h and quantitation of these results (n=4). *P< 0.05 compared with cells treated with NG. (B) Representative Western blots of Sirt6 expression in 30 mM HG-treated podocytes at various times points and quantitation of these results (n=4). *P< 0.05 compared with cells treated with HG for 0 h. (C) Representative Western blots of Sirt6 expression and quantitation of these results in podocytes cultured with different medium (n=4). *P< 0.05 compared with cells in the NG or MA group. (D) Immunofluorescence results of Sirt6 in cultured podocytes in each group(original magnification, ×400). NG=5 mM glucose for 24 h, MA=5 mM glucose+25 mM mannitol for 24 h, HG=30 mM glucose for 24 h.
Figure 6
Figure 6
Effects of HG on mitochondrial morphology and function in cultured podocytes. (A) Representative transmission electron micrographs of mitochondria in cultured podocytes in each group (original magnification, ×8 000, ×15 000). (B) Podocyte mitochondrial numbers were assessed by Mito Green fluorescence staining in different groups (original magnification, ×1 000) and quantitation of these results (n=4). *P< 0.05 compared with cells in the NG or MA group. (C) Mitochondrial oxidative stress and ROS production in podocytes were determined by MitoSox Red fluorescence staining in each group (original magnification, ×1 000) and quantitation of these results (n=4). *P< 0.05 compared with cells in the NG or MA group. (D) Representative images of mitochondrial membrane potential in podocytes by JC-1 staining in different groups (original magnification, ×100) and quantitation of these results.*P< 0.05 compared with cells in the NG or MA group. (E) Western blot results of AMPK and p-AMPK expression in podocytes of different groups and quantitation of these results (n=4).*P< 0.05 compared with cells in the NG or MA group. (F) Western blots detection of cleaved caspase-3 expression in cultured podocytes in each group and quantitation of these results (n=4). *P< 0.05 compared with cells in the NG or MA group. (G) Flow cytometry analysis of apoptosis in cultured podocytes in different groups and quantitation of these results (n=4). *P< 0.05 compared with cells in the NG or MA group. NG=5 mM glucose for 24 h, MA=5 mM glucose+25 mM mannitol for 24 h, HG=30 mM glucose for 24 h.
Figure 7
Figure 7
Sirt6 overexpression attenuated HG induced mitochondrial dysfunction and podocyte injury. Podocytes were transfected with no plasmid, pcDNA3.1, or pcDNA3.1-SIRT6 and then stimulated with HG (30 mM) for 24 h. Untreated and untransfected podocytes were regarded as normal cells. (A) Representative Western blots and quantitative analysis showing the relative protein level of Sirt6 after pcDNA3.1-SIRT6 transfection of podocytes in different groups (n=4). *P< 0.05 compared with normal cells. #P< 0.05 compared with cells treated with HG only. (B) Ultrastructural changes in podocytes by transmission electron microscopy in each group (original magnification, ×8 000, ×15 000). (C) Mitochondrial numbers of podocytes were determined by Mito Green fluorescence staining in different groups (original magnification, ×1 000) and quantitation of these results (n=4). *P< 0.05 compared with normal cells. #P< 0.05 compared with cells treated with HG only. (D) Mitochondrial oxidative stress and ROS production were evaluated by MitoSox Red fluorescence staining in different groups (original magnification, ×1 000) and quantitation of these results (n=4). *P< 0.05 compared with normal cells. #P< 0.05 compared with cells treated with HG only. (E) Representative images of mitochondrial membrane potential of podocytes by JC-1 staining in each group (original magnification, ×100) and quantitation of these results. *P< 0.05 compared with normal cells. #P< 0.05 compared with cells treated with HG only. (F) Western blots detection of AMPK and p-AMPK expression in podocytes for each group and quantitation of these results (n=4). *P< 0.05 compared with normal cells. #P< 0.05 compared with cells treated with HG only. (G) Western blots results of cleaved caspase-3 expression of podocytes in each group and quantitation of these results (n=4). *P< 0.05 compared with normal cells. #P< 0.05 compared with cells treated with HG only. (H) Flow cytometry analysis of apoptosis in the different groups and quantitation of these results (n=4). *P< 0.05 compared with normal cells. #P< 0.05 compared with cells treated with HG only.
Figure 8
Figure 8
A schematic summary of the effects of Sirt6 on HG-induced mitochondrial dysfunction and apoptosis. HG can inhibit Sirt6 expression and induce AMPK dephosphorylation, which contributes to mitochondrial dysfunction in podocytes including increased mitochondrial superoxide production, lower mitochondrial membrane potential and loss of mitochondrial cristae, which finally lead to apoptosis in podocytes. However, Sirt6 overexpression attenuates HG-induced mitochondrial dysfunction and apoptosis via promoting AMPK phosphorylation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Higgins GC, Coughlan MT. Mitochondrial dysfunction and mitophagy: the beginning and end to diabetic nephropathy? Br J Pharmacol. 2014;171:1917–42. - PMC - PubMed
    1. Ahmad J. Management of diabetic nephropathy: Recent progress and future perspective. Diabetes Metab Syndr. 2015;9:343–58. - PubMed
    1. Mathieson PW. The podocyte as a target for therapies-new and old. Nat Rev Nephrol. 2011;8:52–6. - PubMed
    1. Zhou L, Liu Y. Wnt/beta-catenin signalling and podocyte dysfunction in proteinuric kidney disease. Nat Rev Nephrol. 2015;11:535–45. - PMC - PubMed
    1. Burlaka I, Nilsson LM, Scott L, Holtback U, Eklof AC, Fogo AB. et al. Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease. Kidney Int. 2016;90:135–48. - PMC - PubMed

Publication types