Amino acid starvation promotes podocyte autophagy through mammalian target of rapamycin inhibition and transcription factor EB activation

Chen,Yuanhan; Zhao,Xingchen; Li,Jiaxin; Zhang,Li; Li,Ruizhao; Zhang,Hong; Liao,Ruyi; Liu,Shuangxin; Shi,Wei; Liang,Xinling

doi:10.3892/mmr.2018.9438

Amino acid starvation promotes podocyte autophagy through mammalian target of rapamycin inhibition and transcription factor EB activation

Authors:
- Yuanhan Chen
- Xingchen Zhao
- Jiaxin Li
- Li Zhang
- Ruizhao Li
- Hong Zhang
- Ruyi Liao
- Shuangxin Liu
- Wei Shi
- Xinling Liang
View Affiliations

Affiliations: The Second School of Clinical Medicine, Southern Medical University, Guangzhou, Guangdong 510080, P.R. China, Cardiac Surgical Intensive Care Unit, Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080, P.R. China, Division of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangdong Geriatrics Institute, Guangzhou, Guangdong 510080, P.R. China
Published online on: September 3, 2018 https://doi.org/10.3892/mmr.2018.9438
Pages: 4342-4348
Copyright: © Chen 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

Autophagy is important for maintaining normal physiological functions and podocyte cell homeostasis. Amino acid signaling is an important upstream signaling pathway for autophagy regulation. However, the function and the associated mechanism of amino acid signaling in podocyte autophagy is unclear. The present study used normal culture medium and amino acid deprivation medium to culture podocytes in vitro. Multiple methods were utilized to detect autophagic activity including western blot analysis to measure the levels of microtubule‑associated protein 1 light chain 3 (LC3) II and beclin1, reverse transcription‑quantitative polymerase chain reaction was performed to evaluate the levels of LC3 mRNA and transmission electron microscopy was conducted to observe autophagosomes. In addition, tandem green fluorescent protein (GFP)‑monomeric red fluorescent protein (mRFP)‑LC3 adenoviruses were employed to transduce podocytes to observe autophagic flux. Furthermore, the present study examined the effects of amino acid signaling on mammalian target of rapamycin (mTOR) activity and the nuclear translocation of transcription factor EB (TFEB), a core regulator of autophagy, using western blotting and immunofluorescence. The results revealed that amino acid starvation promoted the expression of LC3II and beclin1, and increased the number of autophagosomes and autolysosomes. Amino acid starvation inhibited mTOR activity, and promoted nuclear translocation and TFEB activity. Inhibition of TFEB blocked amino acid starvation‑induced autophagy. These results indicated that amino acid starvation stimulated podocyte autophagy, and thus suggested that mTOR suppression and TFEB activation may mediate amino acid starvation‑induced autophagy in podocytes.

View Figures

View References

1	Mizushima N and Komatsu M: Autophagy: Renovation of cells and tissues. Cell. 147:728–741. 2011. View Article : Google Scholar : PubMed/NCBI
2	Mizushima N, Levine B, Cuervo AM and Klionsky DJ: Autophagy fights disease through cellular self-digestion. Nature. 451:1069–1075. 2008. View Article : Google Scholar : PubMed/NCBI
3	Hartleben B, Gödel M, Meyer-Schwesinger C, Liu S, Ulrich T, Köbler S, Wiech T, Grahammer F, Arnold SJ, Lindenmeyer MT, et al: Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Invest. 120:1084–1096. 2010. View Article : Google Scholar : PubMed/NCBI
4	Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L and Sabatini DM: The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science. 320:1496–1501. 2008. View Article : Google Scholar : PubMed/NCBI
5	Jin G, Lee SW, Zhang X, Cai Z, Gao Y, Chou PC, Rezaeian AH, Han F, Wang CY, Yao JC, et al: Skp2-mediated RagA ubiquitination elicits a negative feedback to prevent amino-acid-dependent mTORC1 hyperactivation by recruiting GATOR1. Mol Cell. 58:989–1000. 2015. View Article : Google Scholar : PubMed/NCBI
6	Settembre C, Di Malta C, Polito VA, Garcia Arencibia M, Vetrini F, Erdin S, Erdin SU, Huynh T, Medina D, Colella P, et al: TFEB links autophagy to lysosomal biogenesis. Science. 332:1429–1433. 2011. View Article : Google Scholar : PubMed/NCBI
7	Settembre C, Zoncu R, Medina DL, Vetrini F, Erdin S, Erdin S, Huynh T, Ferron M, Karsenty G, Vellard MC, et al: A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J. 31:1095–1108. 2012. View Article : Google Scholar : PubMed/NCBI
8	Mundel P, Reiser J, Zúñiga Mejía Borja A, Pavenstädt H, Davidson GR, Kriz W and Zeller R: Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res. 236:248–258. 1997. View Article : Google Scholar : PubMed/NCBI
9	Tan X, Chen Y, Liang X, Yu C, Lai Y, Zhang L, Zhao X, Zhang H, Lin T, Li R and Shi W: Lipopolysaccharide-induced podocyte injury is mediated by suppression of autophagy. Mol Med Rep. 14:811–818. 2016. View Article : Google Scholar : PubMed/NCBI
10	Wei Q and Dong Z: HDAC4 blocks autophagy to trigger podocyte injury: Non-epigenetic action in diabetic nephropathy. Kidney Int. 86:666–668. 2014. View Article : Google Scholar : PubMed/NCBI
11	Zeng C, Fan Y, Wu J, Shi S, Chen Z, Zhong Y, Zhang C, Zen K and Liu Z: Podocyte autophagic activity plays a protective role in renal injury and delays the progression of podocytopathies. J Pathol. 234:203–213. 2014.PubMed/NCBI
12	Inoki K, Mori H, Wang J, Suzuki T, Hong S, Yoshida S, Blattner SM, Ikenoue T, Rüegg MA, Hall MN, et al: mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice. J Clin Invest. 121:2181–2196. 2011. View Article : Google Scholar : PubMed/NCBI
13	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
14	Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, Adachi H, Adams CM, Adams PD, Adeli K, et al: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 12:1–222. 2016. View Article : Google Scholar : PubMed/NCBI
15	Holz MK, Ballif BA, Gygi SP and Blenis J: mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events. Cell. 123:569–580. 2005. View Article : Google Scholar : PubMed/NCBI
16	Zhao X, Chen Y, Tan X, Zhang L, Zhang H, Li Z, Liu S, Li R, Lin T, Liao R, et al: Advanced glycation end-products suppress autophagic flux in podocytes by activating mammalian target of rapamycin and inhibiting nuclear translocation of transcription factor EB. J Pathol. 245:235–248. 2018. View Article : Google Scholar : PubMed/NCBI
17	Yu L, McPhee CK, Zheng L, Mardones GA, Rong Y, Peng J, Mi N, Zhao Y, Liu Z, Wan F, et al: Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature. 465:942–946. 2010. View Article : Google Scholar : PubMed/NCBI
18	Fang L, Li X, Luo Y, He W, Dai C and Yang J: Autophagy inhibition induces podocyte apoptosis by activating the pro-apoptotic pathway of endoplasmic reticulum stress. Exp Cell Res. 322:290–301. 2014. View Article : Google Scholar : PubMed/NCBI
19	Mao N, Tan RZ, Wang SQ, Wei C, Shi XL, Fan JM and Wang L: Ginsenoside Rg1 inhibits angiotensin II-induced podocyte autophagy via AMPK/mTOR/PI3K pathway. Cell Biol Int. 40:917–925. 2016. View Article : Google Scholar : PubMed/NCBI
20	Tagawa A, Yasuda M, Kume S, Yamahara K, Nakazawa J, Chin-Kanasaki M, Araki H, Araki S, Koya D, Asanuma K, et al: Impaired podocyte autophagy exacerbates proteinuria in diabetic nephropathy. Diabetes. 65:755–767. 2016. View Article : Google Scholar : PubMed/NCBI
21	Liu WJ, Li ZH, Chen XC, Zhao XL, Zhong Z, Yang C, Wu HL, An N, Li WY and Liu HF: Blockage of the lysosome-dependent autophagic pathway contributes to complement membrane attack complex-induced podocyte injury in idiopathic membranous nephropathy. Sci Rep. 7:86432017. View Article : Google Scholar : PubMed/NCBI
22	Laidlaw SA, Berg RL, Kopple JD, Naito H, Walker WG and Walser M: Patterns of fasting plasma amino acid levels in chronic renal insufficiency: Results from the feasibility phase of the Modification of Diet in Renal Disease Study. Am J Kidney Dis. 23:504–513. 1994. View Article : Google Scholar : PubMed/NCBI
23	Pena MJ, Lambers Heerspink HJ, Hellemons ME, Friedrich T, Dallmann G, Lajer M, Bakker SJ, Gansevoort RT, Rossing P, de Zeeuw D and Roscioni SS: Urine and plasma metabolites predict the development of diabetic nephropathy in individuals with Type 2 diabetes mellitus. Diabet Med. 31:1138–1147. 2014. View Article : Google Scholar : PubMed/NCBI
24	Kitada M, Ogura Y, Suzuki T, Sen S, Lee SM, Kanasaki K, Kume S and Koya D: A very-low-protein diet ameliorates advanced diabetic nephropathy through autophagy induction by suppression of the mTORC1 pathway in Wistar fatty rats, an animal model of type 2 diabetes and obesity. Diabetologia. 59:1307–1317. 2016. View Article : Google Scholar : PubMed/NCBI