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Deregulation of autophagy under hyperglycemic conditions is dependent on increased lysine 63 ubiquitination: a candidate mechanism in the progression of diabetic nephropathy

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Abstract

Diabetic nephropathy patients (DN) are characterized by increased lysine63 ubiquitination (Lys63-Ub) at the tubular level. Autophagy is deregulated under diabetic conditions, even though the molecular mechanisms and the consequences of this alteration need to be elucidated. The aim of this study was to investigate the link between Lys63-Ub and autophagy in DN and the involvement of these two processes in tubular cell fate. Immunohistochemistry of beclin-1, LC3, and p62 on kidney biopsies highlighted increased protein expression of all these autophagic factors at the tubular level in DN compared to other nephritis. Transmission electron microscopy confirmed the presence of diffuse vacuolization and autophago(lyso)somal structures in proximal tubular cells in DN. Accumulation of Lys63-Ub proteins in DN increased in accordance with the tubular damage and was associated to increased LC3 expression both in vivo and in vitro. Hyperglycemia (HG) induced LC3 and p62 protein expression in HK2 cells together with Lys63-ubiquitinated proteins, and the inhibition of HG-induced Lys63-Ub by NSC697923 inhibitor, significantly reduced both LC3 and p62 expression. Moreover, in DN, those tubules expressing LC3 showed increased caspase-3 expression, supporting the hypothesis that deregulated autophagy induces apoptosis of tubular cells. In vitro, we confirmed a tight association between impaired autophagy, Lys63-Ub, and apoptosis since Lys63-Ub inhibition by NSC697923 abrogated HG-induced cell death and LC3 silencing also blocked hyperglycemia-induced caspase-3 activation. Our data suggested that prolonged hyperglycemia in diabetic patients can impair autophagy as a consequence of Lys63-Ub protein accumulation, thus promoting intracellular autophagic vesicles increase, finally leading to tubular cell death in DN.

Key messages

  • In vivo autophagy is deregulated in diabetic patients with renal disease (DN).

  • Accumulation of Lys63 ubiquitinated proteins is associated to autophagy deregulation.

  • Accumulation of Lys63 ubiquitinated proteins correlated with apoptosis activation.

  • Lys63 ubiquitination inhibition abrogated hyperglycemia-induced autophagy and apoptosis.

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Acknowledgments

We thank Prof. Gianfranco Gennarini, Dr. Picocci Sabrina, and Dr. Antonella Bizzoca of the Dept. of Basic Medical Sciences, Neuroscience and Sense Organs of the University of Bari Aldo Moro for the technical support with confocal microscopy. Dr. Annarita Oranger was supported from 2013 to 2015 as a PhD student in Biotecnologies of Organs transplantations by the University of Bari Aldo Moro. Dr. Mariagrazia Barozzino was supported from 2015 as a PhD student in Biotecnologies of Organs transplantations by the University of Bari Aldo Moro.

Funding

This study was supported by the Ministry of Health (Ricerca finalizzata 2009: 245/RF-2009-1470765).

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Author notes

  1. Paola Pontrelli and Annarita Oranger contributed equally to this work.

Authors and Affiliations

  1. Department of Emergency and Organ Transplantation - Division of Nephrology, University of Bari Aldo Moro, Bari, Italy

    Paola Pontrelli, Annarita Oranger, Mariagrazia Barozzino, Chiara Divella, Francesca Conserva, Massimo Papale, Giuseppe Castellano, Simona Simone & Loreto Gesualdo

  2. Department of Emergency and Organ Transplantation - Division of Pathological Anatomy, University of Bari Aldo Moro, Bari, Italy

    Maria Grazia Fiore, Roberta Rossi & Domenico Piscitelli

  3. Department of Emergency and Organ Transplantation - Division of Endocrinology, University of Bari Aldo Moro, Bari, Italy

    Luigi Laviola & Francesco Giorgino

  4. Department of Basic Medical Sciences, Neurosciences and Sense Organs - Division of Applied Biology, University of Bari Aldo Moro, Bari, Italy

    Anna Gallone

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  1. Paola Pontrelli
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Correspondence to Paola Pontrelli.

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The study was conducted according to the last version of the Declaration of Helsinki and was approved by the local ethics committee (Prot. N.4104/2013).

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The authors declare that they have no conflict of interest.

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Pontrelli, P., Oranger, A., Barozzino, M. et al. Deregulation of autophagy under hyperglycemic conditions is dependent on increased lysine 63 ubiquitination: a candidate mechanism in the progression of diabetic nephropathy. J Mol Med 96, 645–659 (2018). https://doi.org/10.1007/s00109-018-1656-3

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  • DOI: https://doi.org/10.1007/s00109-018-1656-3

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