Abstract
Insufficient autophagy in podocytes is related to podocyte injury in diabetic nephropathy (DN). Advanced glycation end-products (AGEs) are major factors of podocyte injury in DN. However, the role and mechanism of AGEs in autophagic dysfunction remain unknown. We investigated autophagic flux in AGE-stimulated cultured podocytes using multiple assays: western blotting, reverse transcription-quantitative PCR, immunofluorescence staining, and electron microscopy. We also utilized chloroquine and a fluorescent probe to monitor the formation and turnover of autophagosomes. Mice of the db/db strain were used to model diabetes mellitus (DM) with high levels of AGEs. To mimic DM with normal levels of AGEs as a control, we treated db/db mice with pyridoxamine to block AGE formation. AGEs impaired autophagic flux in the cultured podocytes. Compared with db/db mice with normal AGEs but high glucose levels, db/db mice with high AGEs and high glucose levels exhibited lower autophagic activity. Aberrant autophagic flux was related to hyperactive mammalian target of rapamycin (mTOR), a major suppressor of autophagy. Pharmacologic inhibition of mTOR activity restored impaired autophagy. AGEs inhibited the nuclear translocation and activity of the pro-autophagic transcription factor EB (TFEB) and thus suppressed transcription of its several autophagic target genes. Conversely, TFEB overexpression prevented AGE-induced autophagy insufficiency. Attenuating mTOR activity recovered TFEB nuclear translocation under AGE stimulation. Co-immunoprecipitation assays further demonstrated the interaction between mTOR and TFEB in AGE-stimulated podocytes and in glomeruli from db/db mice. In conclusion, AGEs play a crucial part in suppressing podocyte autophagy under DM conditions. AGEs inhibited the formation and turnover of autophagosomes in podocytes by activating mTOR and inhibiting the nuclear translocation of TFEB. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.