Impaired Podocyte Autophagy Exacerbates Proteinuria in Diabetic Nephropathy

Tagawa, Atsuko; Yasuda, Mako; Kume, Shinji; Yamahara, Kosuke; Nakazawa, Jun; Chin‐Kanasaki, Masami; Araki, Hisazumi; Araki, Shin‐ichi; Koya, Daisuke; Asanuma, Katsuhiko; Kim, Eun‐Hee; Haneda, Masakazu; Kajiwara, Nobuyuki; Hayashi, Kazuyuki; Ôhashi, Hiroshi; Ugi, Satoshi; Maegawa, Hiroshi; Uzu, Takashi

doi:10.2337/db15-0473

Cited by 255 publications

(219 citation statements)

References 52 publications

(69 reference statements)

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“…We also found a decreased number of autolysosomes under AGE stimulation using GFP‐mRFP‐LC3 probes. This impaired autophagosome turnover was supported by the decreased transcript levels for the lysosomal gene Lamp1 and is consistent with Tagawa et al 's report 24. Therefore, AGEs disrupted multiple steps of autophagic flux in podocytes.…”

Section: Discussionsupporting

confidence: 90%

Advanced glycation end‐products suppress autophagic flux in podocytes by activating mammalian target of rapamycin and inhibiting nuclear translocation of transcription factor EB

Zhao

Chen

Tan

et al. 2018

The Journal of Pathology

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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.

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Section: Discussionsupporting

confidence: 90%

Advanced glycation end‐products suppress autophagic flux in podocytes by activating mammalian target of rapamycin and inhibiting nuclear translocation of transcription factor EB

Zhao

Chen

Tan

et al. 2018

The Journal of Pathology

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“…In this in vitro study, we found that podocyte injury induced by PAN was associated with autophagy activity. Autophagy levels were found to be negatively correlated with mTOR activation in injured podocytes, and the mTOR-autophagy balance of low-level mTOR and high-level autophagy was uncoupled, consistent with previous reports [1,20]; however, we also observed that RAP could increase the level of autophagy and reduce podocyte apoptosis by inhibiting the mTOR/P70S6K/4EBP1 signaling pathway. These data strongly indicate that autophagy plays a major role in maintaining podocyte homeostasis and protects damaged podocytes.…”

Section: Discussionsupporting

confidence: 81%

Rapamycin Reduces Podocyte Apoptosis and is Involved in Autophagy and mTOR/ P70S6K/4EBP1 Signaling

Jin

et al. 2018

Cell Physiol Biochem

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Background/Aims: The purpose of this study was to investigate the impact of rapamycin (RAP) on autophagy in podocytes and the therapeutic effects of RAP on idiopathic membranous nephropathy (IMN). Methods: We established an in vitro model of IMN by preconditioning mouse podocytes with puromycin aminonucleoside (PAN). A Cell Counting Kit-8 was used to detect the proliferation of each group of podocytes. Podocyte apoptosis was analyzed by flow cytometry via annexin V/propidium iodide dual staining. Subsequently, we observed the number of autophagosomes by transmission electron microscopy. Western blotting was used to detect the levels of LC3, mTOR, p-mTOR, 4EBP1, p-4EBP1, P70S6K, and p-P70S6K in each group. Results: The number of podocytes in the PAN + 100 ng/mL RAP group, PAN + 200 ng/mL RAP group, and PAN + 300 ng/mL RAP group was significantly increased (P < 0.01). The apoptotic rate of podocytes was significantly different between the PAN group and the PAN + RAP group (P < 0.001). There were fewer autophagic corpuscles in the PAN group and more autophagosomes were observed in the PAN + RAP group. LC3 protein expression was down-regulated in the PAN group, while its expression was up-regulated in the PAN + RAP group. In the PAN group, the levels of phosphorylated mTOR, 4EBP1, and P70S6K were increased, while in the PAN + RAP group, protein phosphorylation was reduced. Conclusions: RAP can effectively inhibit the mTOR/P70S6K/4EBP1 signaling pathway, and activate podocyte autophagy, consequently reducing podocyte apoptosis. Therefore, RAP could be used for the treatment of idiopathic membranous nephropathy.

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“…However, the lossy or excessive autophagy also has the capacity to cause abnormal conditions. It has been reported that insufficient podocyte autophagy was involved in the pathogenesis of podocytes loss and led to massive proteinuria by high-fat diet in Atg5 deficient mice26. Contradictory excessive autophagy in adipocytes caused insulin resistance of adipose tissue in obese patients with type 2 diabetes27.…”

Section: Discussionmentioning

confidence: 99%

AGEs Induced Autophagy Impairs Cutaneous Wound Healing via Stimulating Macrophage Polarization to M1 in Diabetes

Guo

Cai

et al. 2016

Sci Rep

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Autophagy is essential in physiological and pathological processes, however, the role of autophagy in cutaneous wound healing and the underlying molecular mechanism remain elusive. We hypothesized that autophagy plays an important role in regulating wound healing. Here, we show that enhanced autophagy negatively impacts on normal cutaneous healing process and is related to chronic wounds as demonstrated by the increased LC3 in diabetic mice skin or patients’ chronic wounds. In addition, inhibition of autophagy by 3-MA restores delayed healing in C57BL/6 or db/db mice, demonstrating that autophagy is involved in regulating wound healing. Furthermore, we identify that macrophage is a major cell type underwent autophagy in wounds and increased autophagy induces macrophages polarization into M1 with elevated CD11c population and gene expressions of proinflammatory cytokines. To explore the mechanism underlying autophagy-impaired wound healing, we tested the role of IRF8, a regulator of autophagy, in autophagy-modulated macrophages polarization. IRF8 activation is up-regulating autophagy and M1 polarization of macrophages after AGEs (advanced glycation endproducts) treatment, blocking the IRF8 with shIRF8 inhibits autophagic activity and M1 polarization. In summary, this study elucidates that AGEs induces autophagy and modulates macrophage polarization to M1 via IRF8 activation in impairment of cutaneous wound healing.

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Impaired Podocyte Autophagy Exacerbates Proteinuria in Diabetic Nephropathy

Cited by 255 publications

References 52 publications

Advanced glycation end‐products suppress autophagic flux in podocytes by activating mammalian target of rapamycin and inhibiting nuclear translocation of transcription factor EB

Advanced glycation end‐products suppress autophagic flux in podocytes by activating mammalian target of rapamycin and inhibiting nuclear translocation of transcription factor EB

Rapamycin Reduces Podocyte Apoptosis and is Involved in Autophagy and mTOR/ P70S6K/4EBP1 Signaling

AGEs Induced Autophagy Impairs Cutaneous Wound Healing via Stimulating Macrophage Polarization to M1 in Diabetes

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