Department of Nephrology, Ajou University School of Medicine, Suwon, Korea.
PLoS One. 2019 Feb 22;14(2):e0212818. doi: 10.1371/journal.pone.0212818. eCollection 2019.
Growth Arrest and DNA Damage 45γ (GADD45γ) is a member of the DNA damage-inducible gene family which responds to environmental stresses. Apoptosis is a critical mode of renal tubular cell death in nephrotoxin-induced acute kidney injury. In this study, we investigated the role of GADD45γ in renal tubular cell apoptosis induced by nephrotoxic drugs.
Primary human renal tubular epithelial (HRE) cells were used in this study. To derive stable cell lines in which GADD45γ expression was silenced, HRE cells were transduced with a plasmid encoding GADD45γ-specific shRNA. The recombinant adenovirus containing the GADD45γ gene was synthesized to overexpress GADD45γ protein. Cell death was induced by cisplatin and cyclosporine A (CsA). To prevent apoptotic cell death, pan-caspase inhibitor ZVAD-FMK was used. To prevent non-apoptotic cell death, necrostatin-1 and ferrostatin-1 were used. The degree of apoptosis and necrosis of cultured cells were evaluated by flow cytometry.
Expression of the GADD45γ gene was significantly upregulated in response to treatment with CsA and cisplatin. Apoptosis and necrosis induced by these drugs were significantly reduced by silencing of GADD45γ, and significantly augmented by the overexpression of GADD45γ. The activation of caspase-3 and caspase-7 as well as caspase-9 induced by cisplatin or CsA was reduced by silencing of GADD45γ, and was augmented by the overexpression of GADD45γ, indicating that caspase activation is dependent on the expression of GADD45γ. ZVAD-FMK significantly inhibited apoptosis induced by cisplatin or CsA, indicating a role of caspases in mediating apoptotic cell death. ZVAD-FMK was effective to prevent necrosis as well, indicating that the observed necrosis was a secondary event following apoptosis at least in part.
To our knowledge, this is the first study to show that GADD45γ is required for the caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.
生长停滞和 DNA 损伤 45γ（GADD45γ）是一种 DNA 损伤诱导基因家族的成员，对环境应激作出反应。细胞凋亡是肾毒物诱导急性肾损伤中肾小管细胞死亡的关键方式。在这项研究中，我们研究了 GADD45γ 在肾毒物诱导的肾小管细胞凋亡中的作用。
本研究使用原代人肾小管上皮（HRE）细胞。为了沉默 GADD45γ 的表达，将 HRE 细胞转导携带 GADD45γ 特异性 shRNA 的质粒。合成含有 GADD45γ 基因的重组腺病毒以过表达 GADD45γ 蛋白。用顺铂和环孢素 A（CsA）诱导细胞死亡。用泛半胱天冬酶抑制剂 ZVAD-FMK 预防细胞凋亡性死亡，用坏死抑制剂 1（necrostatin-1）和铁死亡抑制剂 1（ferrostatin-1）预防非凋亡性死亡。用流式细胞术评估培养细胞的凋亡和坏死程度。
GADD45γ 基因的表达在 CsA 和顺铂处理后显著上调。用 GADD45γ 沉默抑制这些药物诱导的凋亡和坏死，用 GADD45γ 过表达增强这些药物诱导的凋亡和坏死。CsA 或顺铂诱导的 caspase-3 和 caspase-7 以及 caspase-9 的激活被 GADD45γ 沉默抑制，被 GADD45γ 过表达增强，表明 caspase 激活依赖于 GADD45γ 的表达。ZVAD-FMK 显著抑制顺铂或 CsA 诱导的凋亡，表明 caspase 在介导细胞凋亡性死亡中起作用。ZVAD-FMK 对预防坏死也有效，表明观察到的坏死至少部分是凋亡后的继发事件。
据我们所知，这是第一项表明 GADD45γ 是肾毒物诱导肾小管细胞 caspase 依赖性凋亡所必需的研究。
