Mitochondria-associated endoplasmic reticulum membranes (MAMs) regulate ATG14- and Beclin1-mediated mitophagy and play key roles in the development of diabetic nephropathy (DN). DsbA-L is mainly located in MAMs and plays a role in renoprotection, but whether it activates mitophagy by maintaining MAM integrity remains unclear. In the present study, we found that renal tubular damage was further aggravated in diabetic DsbA-L-/- mice compared with diabetic mice and that this damage was accompanied by disrupted MAM integrity and decreased mitophagy. Furthermore, notably decreased expression of ATG14 and Beclin1 in MAMs extracted from the kidneys of diabetic DsbA-L-/- mice was observed. In vitro, overexpression of DsbA-L reversed the disruption of MAM integrity and enhanced mitophagy in HK-2 cells, a human proximal tubular cell line, after exposure to high-glucose (HG) conditions. Additionally, compared with control mice, DsbA-L-/- mice were exhibited down-regulated expression of helicase with zinc finger 2 (HELZ2) in their kidneys according to transcriptome analysis; HELZ2 serves as a cotranscription factor that synergistically functions with PPARα to promote the expression of mitofusin 2 (MFN-2). Treatment of HK-2 cells with MFN-2 siRNA resulted in MAM uncoupling and decreased mitophagy. Moreover, HG notably reduced the expression of HELZ2 and MFN-2 and inhibited mitophagy, and these effects were partially blocked by overexpression of DsbA-L and altered upon cotreatment with HELZ2 siRNA, HELZ2 overexpression or MK886 (PPARα inhibitor) treatment. These data indicate that DsbA-L alleviates diabetic tubular damage by activating mitophagy through maintenance of MAM integrity via the HELZ2/MFN-2 pathway.

中文翻译：

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DsbA-L 通过维持 MAM 完整性来激活线粒体自噬，从而减轻糖尿病肾病的肾小管损伤。

线粒体相关内质网膜 (MAM) 调节 ATG14 和 Beclin1 介导的线粒体自噬，并在糖尿病肾病 (DN) 的发展中发挥关键作用。DsbA-L 主要位于 MAM 中，在肾脏保护中发挥作用，但其是否通过维持 MAM 完整性来激活线粒体自噬仍不清楚。在本研究中，我们发现与糖尿病小鼠相比，糖尿病DsbA-L-/-小鼠的肾小管损伤进一步加剧，并且这种损伤伴随着MAM完整性的破坏和线粒体自噬的减少。此外，从糖尿病 DsbA-L-/- 小鼠肾脏提取的 MAM 中观察到 ATG14 和 Beclin1 的表达显着降低。在体外，DsbA-L 的过度表达逆转了 MAM 完整性的破坏并增强了 HK-2 细胞（一种人近端肾小管细胞系）的线粒体自噬，暴露于高血糖（HG）条件后。此外，根据转录组分析，与对照小鼠相比，DsbA-L-/-小鼠肾脏中锌指解旋酶2（HELZ2）的表达下调；HELZ2 作为共转录因子，与 PPARα 协同作用，促进线粒体融合蛋白 2 (MFN-2) 的表达。用 MFN-2 siRNA 处理 HK-2 细胞导致 MAM 解偶联并减少线粒体自噬。此外，HG 显着降低 HELZ2 和 MFN-2 的表达并抑制线粒体自噬，这些作用被 DsbA-L 的过表达部分阻断，并在与 HELZ2 siRNA、HELZ2 过表达或 MK886（PPARα 抑制剂）共同治疗时发生改变。