Small GTP-binding protein GDP dissociation stimulator influences cisplatin-induced acute kidney injury via PERK-dependent ER stress

Cisplatin is a common anticancer drug, but its frequent nephrotoxicity limits its clinical use. Small GTP-binding protein GDP dissociation stimulator (smgGDS), a small GTPase chaperone protein, was considerably downregulated during cisplatin-induced acute kidney injury (CDDP-AKI), especially in renal tubular epithelial cells. SmgGDS-knockdown mice was established and found that smgGDS knockdown promoted CDDP-AKI, as demonstrated by an increase in serum creatine, blood urea nitrogen levels and the appearance of tubular patterns. RNA sequencing suggested that protein kinase RNA-like ER kinase (PERK), which bridges mitochondria-associated ER membranes, was involved in smgGDS knockdown following CDDP-AKI, and then identified that smgGDS knockdown increased phosphorylated-PERK in vivo and in vitro. Furthermore, we confirmed that smgGDS deficiency aggravated apoptosis and ER stress in vivo and in vitro. And the ER stress inhibitor 4-Phenylbutyric acid and the inhibition of PERK phosphorylation mitigated smgGDS deficiency-induced ER stress related apoptosis following cisplatin treatment, while the eIF2α phosphorylation inhibitor could not reverse the smgGDS deficiency accelerated cell death. Furthermore, the over-expression of smgGDS could reverse the ER stress and apoptosis caused by CDDP. Overall, smgGDS regulated PERK-dependent ER stress and apoptosis, thereby influencing renal damage. This study identified a target for diagnosing and treating cisplatin-induced acute kidney injury. The smgGDS-knockdown cell line was established to explore the underlying mechanism of CDDP-AKI, and shRNA targeting smgGDS produced a robust knockdown of smgGDS protein expression compared with control shRNA. For further mechanistic studies, the RNA-seq and related bioinformatics analysis were performed.

顺铂（cisplatin）是临床常用的抗肿瘤药物，但其频发的肾毒性限制了其临床应用。小GTP结合蛋白GDP解离刺激因子（small GTP-binding protein GDP dissociation stimulator，smgGDS）作为一种小GTP酶伴侣蛋白，在顺铂诱导的急性肾损伤（cisplatin-induced acute kidney injury，CDDP-AKI）进程中显著下调，尤其在肾小管上皮细胞中下调更为显著。研究人员构建了smgGDS敲低小鼠模型，发现敲低smgGDS可加重CDDP-AKI，具体表现为血清肌酐、血尿素氮水平升高，以及肾小管损伤病理征象的出现。RNA测序（RNA sequencing）结果显示，蛋白激酶RNA样内质网激酶（protein kinase RNA-like ER kinase，PERK）作为连接线粒体相关内质网膜的关键分子，参与了CDDP-AKI背景下smgGDS敲低介导的病理过程；后续研究证实，smgGDS敲低可在体内外上调磷酸化PERK的表达水平。此外，本研究证实smgGDS缺失可在体内外加剧细胞凋亡与内质网应激（endoplasmic reticulum stress，ER stress）。内质网应激抑制剂4-苯基丁酸（4-Phenylbutyric acid）与PERK磷酸化抑制剂均可减轻顺铂处理后smgGDS缺失诱导的内质网应激相关细胞凋亡；而真核翻译起始因子2α（eukaryotic translation initiation factor 2α，eIF2α）磷酸化抑制剂则无法逆转smgGDS缺失加速的细胞死亡。此外，过表达smgGDS可逆转CDDP诱导的内质网应激与细胞凋亡。综上，smgGDS可通过调控PERK依赖的内质网应激与细胞凋亡过程，进而影响肾脏损伤程度。本研究明确了顺铂诱导急性肾损伤的诊断与治疗靶点。为探究CDDP-AKI的潜在发病机制，本研究构建了smgGDS敲低细胞系；与阴性对照短发夹RNA（short hairpin RNA，shRNA）相比，靶向smgGDS的shRNA可显著抑制smgGDS的蛋白表达。为开展后续机制研究，本研究进行了RNA测序（RNA sequencing，RNA-seq）及相关生物信息学分析。