Sirt3 promotes the autophagy of HK‑2 human proximal tubular epithelial cells via the inhibition of Notch‑1/Hes‑1 signaling

Wang,Ying; Chang,Jiang; Wang,Zi-Qiang; Li,Ying

doi:10.3892/mmr.2021.12273

Sirt3 promotes the autophagy of HK‑2 human proximal tubular epithelial cells via the inhibition of Notch‑1/Hes‑1 signaling

Authors:
- Ying Wang
- Jiang Chang
- Zi-Qiang Wang
- Ying Li
View Affiliations

Affiliations: Department of Nephrology, Bayan Nur Hospital, Bayan Nur, Inner Mongolia Autonomous Region 015000, P.R. China, Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Hainan Medical College, Haikou, Hainan 570102, P.R. China, Department of Nephrology, The Third Hospital of Hebei Medical University, Shijiazhuang, Hebei 050051, P.R. China
Published online on: July 6, 2021 https://doi.org/10.3892/mmr.2021.12273
Article Number: 634
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Diabetic nephropathy (DN) is a predominant cause of end‑stage renal disease. The impairment of the autophagy of human renal tubular epithelial cells (HK‑2 cells) is involved in the pathogenic mechanisms of DN. Sirtuin (Sirt)3 regulates the scavenging of damaged organelles and maintains energy balance. The present study aimed to examine the protective effects of Sirt3 on HK‑2 cells stimulated by high glucose (HG). HK‑2 cells were cultured in normal glucose (NG), HG or hyperosmotic medium. The viability of the HK‑2 cells was detected using a Cell Counting Kit‑8 assay. The expression and localization of Sirt3 were detected via immunofluorescence. Following transfection with an overexpression plasmid, the expression levels of key components in the Notch homolog 1 (Notch‑1)/hairy and enhancer of split‑1 (Hes‑1) pathway and those of the autophagy‑related proteins, Beclin‑1, LC‑3II and p62, were measured by western blot analysis and reverse transcription‑quantitative PCR (RT‑qPCR). As the Notch‑1/Hes‑1 pathway was inhibited, the expression levels of Beclin‑1, LC‑3II and p62 were also examined at transcriptional and translational level. It was found that prolonged culture in HG medium markedly reduced cell viability compared with the cells cultured in NG or in NG + mannitol, an effect that was aggravated with the increasing duration of culture. HG was capable of inhibiting the expression levels of Beclin‑1, LC‑3II and Sirt3, and upregulating p62 and the Notch‑1/Hes‑1 pathway, as verified by western blot analysis and RT‑qPCR. The results of immunofluorescence staining revealed that HG decreased Sirt3 expression. Sirt3 reversed the HG‑induced inhibition of the expression of Beclin‑1 and LC‑3II and the upregulation of p62. Moreover, Sirt3 reversed the HG‑induced inhibition of the Notch‑1/Hes‑1 signaling pathway. However, this autophagy‑promoting effect of Sirt3 was counteracted by the Notch‑1/Hes‑1 pathway activator. On the whole, the present study demonstrated that Sirt3 promoted the autophagy of HK‑2 cells, at least partly, via the downregulation of Notch‑1/Hes‑1.

View Figures

View References

1	Warren AM, Knudsen ST and Cooper ME: Diabetic nephropathy: An insight into molecular mechanisms and emerging therapies. Expert Opin Ther Targets. 23:579–591. 2019. View Article : Google Scholar : PubMed/NCBI
2	Gilbertson DT, Liu J, Xue JL, Louis TA, Solid CA, Ebben JP and Collins AJ: Projecting the number of patients with end-stage renal disease in the United States to the year 2015. J Am Soc Nephrol. 16:3736–3741. 2005. View Article : Google Scholar : PubMed/NCBI
3	Tang C, Livingston MJ, Liu Z and Dong Z: Autophagy in kidney homeostasis and disease. Nat Rev Nephrol. 16:489–508. 2020. View Article : Google Scholar : PubMed/NCBI
4	Small DM, Bennett NC, Coombes J, Johnson DW and Gobe GC: Mitochondrial homeostasis is impeded by degradation and autophagy in oxidative stress-induced renal cell injury. Revista Española De Reumatismo Y Enfermedades Osteoarticulares. 11:67–73. 2013.
5	Kitada M, Ogura Y, Monno I and Koya D: Regulating autophagy as a therapeutic target for diabetic nephropathy. Curr Diab Rep. 17:532017. View Article : Google Scholar : PubMed/NCBI
6	Zhu SY, Yao RQ, Li YX, Zhao PY, Ren C, Du XH and Yao YM: Lysosomal quality control of cell fate: A novel therapeutic target for human diseases. Cell Death Dis. 11:8172020. View Article : Google Scholar : PubMed/NCBI
7	Guo J, Zheng HJ, Zhang W, Lou W, Xia C, Han XT, Huang WJ, Zhang F, Wang Y and Liu WJ: Accelerated kidney aging in diabetes mellitus. Oxid Med Cell Longev. 2020:12340592020. View Article : Google Scholar : PubMed/NCBI
8	Lee IH, Cao L, Mostoslavsky R, Lombard DB, Liu J, Bruns NE, Tsokos M, Alt FW and Finkel T: A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. Proc Natl Acad Sci USA. 105:3374–3379. 2008. View Article : Google Scholar : PubMed/NCBI
9	Li R, Xin T, Li D, Wang C, Zhu H and Zhou H: Therapeutic effect of Sirtuin 3 on ameliorating nonalcoholic fatty liver disease: The role of the ERK-CREB pathway and Bnip3-mediated mitophagy. Redox Biol. 18:229–243. 2018. View Article : Google Scholar : PubMed/NCBI
10	Zhang T, Liu J, Shen S, Tong Q, Ma X and Lin L: SIRT3 promotes lipophagy and chaperon-mediated autophagy to protect hepatocytes against lipotoxicity. Cell Death Differ. 27:329–344. 2020. View Article : Google Scholar : PubMed/NCBI
11	Zhao W, Zhang L, Chen R, Lu H, Sui M, Zhu Y and Zeng L: SIRT3 protects against acute kidney injury via AMPK/mTOR-regulated autophagy. Front Physiol. 9:15262018. View Article : Google Scholar : PubMed/NCBI
12	Kitada M, Kume S, Takeda-Watanabe A, Kanasaki K and Koya D: Sirtuins and renal diseases: Relationship with aging and diabetic nephropathy. Clin Sci (Lond). 124:153–164. 2013. View Article : Google Scholar : PubMed/NCBI
13	Yu W, Gao B, Li N, Wang J, Qiu C, Zhang G, Liu M, Zhang R, Li C, Ji G and Zhang Y: Sirt3 deficiency exacerbates diabetic cardiac dysfunction: Role of Foxo3A-Parkin-mediated mitophagy. Biochim Biophys Acta Mol Basis Dis. 1863:1973–1983. 2017. View Article : Google Scholar : PubMed/NCBI
14	Chen Y, Zhang F, Wang D, Li L, Si H, Wang C, Liu J, Chen Y, Cheng J and Lu Y: Mesenchymal stem cells attenuate diabetic lung fibrosis via adjusting Sirt3-mediated stress responses in rats. Oxid Med Cell Longev. 2020:80761052020.PubMed/NCBI
15	Artavanis-Tsakonas S, Rand MD and Lake RJ: Notch signaling: Cell fate control and signal integration in development. Science. 284:770–776. 1999. View Article : Google Scholar : PubMed/NCBI
16	Yuri S, Nishikawa M and Yanagawa N, Jo OD and Yanagawa N: Maintenance of mouse nephron progenitor cells in aggregates with Gamma-secretase inhibitor. PLoS One. 10:e01292422015. View Article : Google Scholar : PubMed/NCBI
17	Wang Y, Li Y, Yang Z, Wang Z, Chang J, Zhang T, Chi Y, Han N and Zhao K: Pyridoxamine treatment of HK-2 human proximal tubular epithelial cells reduces oxidative stress and the inhibition of autophagy induced by high glucose levels. Med Sci Monit. 25:1480–1488. 2019. View Article : Google Scholar : PubMed/NCBI
18	Jiao X, Li Y, Zhang T, Liu M and Chi Y: Role of Sirtuin3 in high glucose-induced apoptosis in renal tubular epithelial cells. Biochem Biophys Res Commun. 480:387–393. 2016. View Article : Google Scholar : PubMed/NCBI
19	Wang Z, Li Y, Wang Y, Zhao K, Chi Y and Wang B: Pyrroloquinoline quinine protects HK-2cells against high glucose-induced oxidative stress and apoptosis through Sirt3 and PI3K/Akt/FoxO3a signaling pathway. Biochem Biophys Res Commun. 508:398–404. 2019. View Article : Google Scholar : PubMed/NCBI
20	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
21	Mizushima N and Levine B: Autophagy in human diseases. N Engl J Med. 383:1564–1576. 2020. View Article : Google Scholar : PubMed/NCBI
22	Jiang P and Mizushima N: Autophagy and human diseases. Cell Res. 24:69–79. 2014. View Article : Google Scholar : PubMed/NCBI
23	Kim KH and Lee MS: Autophagy-a key player in cellular and body metabolism. Nat Rev Endocrinol. 10:322–337. 2014. View Article : Google Scholar : PubMed/NCBI
24	Ding Y and Choi ME: Autophagy in diabetic nephropathy. J Endocrinol. 224:R15–R30. 2015. View Article : Google Scholar : PubMed/NCBI
25	Liu WJ, Huang WF, Ye L, Chen RH, Yang C, Wu HL, Pan QJ and Liu HF: The activity and role of autophagy in the pathogenesis of diabetic nephropathy. Eur Rev Med Pharmacol Sci. 22:3182–3189. 2018.PubMed/NCBI
26	Lin F: Autophagy in renal tubular injury and repair. Acta Physiol (Oxf). 220:229–237. 2017. View Article : Google Scholar : PubMed/NCBI
27	Ng F and Tang BL: Sirtuins' modulation of autophagy. J Cell Physiol. 228:2262–2270. 2013. View Article : Google Scholar : PubMed/NCBI
28	Peng Y, Yang C, Shi X, Li L, Dong H, Liu C, Fang Z, Wang Z, Ming S, Liu M, et al: Sirt3 suppresses calcium oxalate-induced renal tubular epithelial cell injury via modification of FoxO3a-mediated autophagy. Cell Death Dis. 10:342019. View Article : Google Scholar : PubMed/NCBI
29	Zhang M, Deng YN, Zhang JY, Liu J, Li YB, Su H and Qu QM: SIRT3 protects Rotenone-induced injury in SH-SY5Y cells by promoting autophagy through the LKB1-AMPK-mTOR Pathway. Aging Dis. 9:273–286. 2018. View Article : Google Scholar : PubMed/NCBI
30	Wang Y, Zhang X, Wang P, Shen Y, Yuan K, Li M, Liang W and Que H: Sirt3 overexpression alleviates hyperglycemia-induced vascular inflammation through regulating redox balance, cell survival, and AMPK-mediated mitochondrial homeostasis. J Recept Signal Transduct Res. 39:341–349. 2019. View Article : Google Scholar : PubMed/NCBI
31	Surendran K, Boyle S, Barak H, Kim M, Stomberski C, McCright B and Kopan R: The contribution of Notch1 to nephron segmentation in the developing kidney is revealed in a sensitized Notch2 background and can be augmented by reducing Mint dosage. Dev Biol. 337:386–395. 2010. View Article : Google Scholar : PubMed/NCBI
32	Vooijs M, Ong CT, Hadland B, Huppert S, Liu Z, Korving J, van den Born M, Stappenbeck T, Wu Y, Clevers H and Kopan R: Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE. Development. 134:535–544. 2007. View Article : Google Scholar : PubMed/NCBI
33	Zheng D, Tao M, Liang X, Li Y, Jin J and He Q: p66Shc regulates podocyte autophagy in high glucose environment through the Notch-PTEN-PI3K/Akt/mTOR pathway. Histol Histopathol. 35:405–415. 2020.PubMed/NCBI