Department of Emergency and Organ Transplantation - Division of Nephrology, University of Bari Aldo Moro, Bari, Italy.
Department of Emergency and Organ Transplantation - Division of Pathological Anatomy, University of Bari Aldo Moro, Bari, Italy.
J Mol Med (Berl). 2018 Jul;96(7):645-659. doi: 10.1007/s00109-018-1656-3. Epub 2018 May 27.
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.
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.
糖尿病肾病（DN）患者的肾小管水平存在赖氨酸 63 泛素化（Lys63-Ub）增加。尽管需要阐明这种改变的分子机制和后果，但在糖尿病条件下，自噬受到了调节。本研究的目的是研究 DN 中 Lys63-Ub 与自噬之间的联系，以及这两个过程在肾小管细胞命运中的作用。对肾脏活检进行 beclin-1、LC3 和 p62 的免疫组化分析表明，与其他肾炎相比，DN 肾小管中所有这些自噬因子的蛋白表达均增加。透射电镜证实 DN 中近端肾小管细胞存在弥漫性空泡化和自噬体（溶酶体）结构。DN 中 Lys63-Ub 蛋白的积累与肾小管损伤一致，并与体内和体外 LC3 表达增加相关。高血糖（HG）诱导 HK2 细胞中 LC3 和 p62 蛋白表达以及 Lys63-泛素化蛋白表达，并用 NSC697923 抑制剂抑制 HG 诱导的 Lys63-Ub 可显著降低 LC3 和 p62 的表达。此外，在 DN 中，表达 LC3 的那些肾小管中 caspase-3 表达增加，支持自噬失调导致肾小管细胞凋亡的假说。在体外，我们证实了自噬受损、Lys63-Ub 和细胞凋亡之间存在紧密联系，因为 NSC697923 抑制 Lys63-Ub 可阻断 HG 诱导的细胞死亡，LC3 沉默也可阻断高血糖诱导的 caspase-3 激活。我们的数据表明，糖尿病患者的长期高血糖可能会导致 Lys63-Ub 蛋白积累，从而损害自噬，从而促进细胞内自噬小泡增加，最终导致 DN 中肾小管细胞死亡。
体内自噬在患有肾脏疾病（DN）的糖尿病患者中失调。Lys63 泛素化蛋白的积累与自噬失调有关。Lys63 泛素化蛋白的积累与凋亡激活相关。Lys63 泛素化抑制可阻断高血糖诱导的自噬和凋亡。
