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Amino acid starvation promotes podocyte autophagy through mammalian target of rapamycin inhibition and transcription factor EB activation.
Molecular Medicine Reports 2018 November
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.
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