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Growth arrest and DNA damage 45? is required for caspase-dependent renal tubular cell apoptosis.

ABSTRACT: BACKGROUND:Growth Arrest and DNA Damage 45? (GADD45?) 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 GADD45? in renal tubular cell apoptosis induced by nephrotoxic drugs. METHODS:Primary human renal tubular epithelial (HRE) cells were used in this study. To derive stable cell lines in which GADD45? expression was silenced, HRE cells were transduced with a plasmid encoding GADD45?-specific shRNA. The recombinant adenovirus containing the GADD45? gene was synthesized to overexpress GADD45? 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. RESULTS:Expression of the GADD45? 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 GADD45?, and significantly augmented by the overexpression of GADD45?. The activation of caspase-3 and caspase-7 as well as caspase-9 induced by cisplatin or CsA was reduced by silencing of GADD45?, and was augmented by the overexpression of GADD45?, indicating that caspase activation is dependent on the expression of GADD45?. 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. CONCLUSIONS:To our knowledge, this is the first study to show that GADD45? is required for the caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.

SUBMITTER: Shin GT

PROVIDER: S-EPMC6386268 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Growth arrest and DNA damage 45γ is required for caspase-dependent renal tubular cell apoptosis.

Shin Gyu-Tae GT Lee Hwa Joung HJ Park Ji Eun JE

PloS one 20190222 2

<h4>Background</h4>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.<h4>Methods</h4>Primary human renal tubular epithelial (HRE) cells were used in this study. To derive stable cell lines in which GA ...[more]

PMID: 30794682

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Project description:BackgroundAcute kidney injury (AKI) is a common clinically critical illness with serious consequences for the patients. Shenshuaikang enema (SE) is a Chinese herbal compound that is used to treat AKI in clinical practice. However, its mechanism of action remains unclear.AimThe aim of this study was to investigate the therapeutic effect of SE and explore the molecular mechanisms using network pharmacology and in vitro experiments.Materials and methodsThe herb-component-target network was constructed based on network pharmacology. The predicted targets and pathways were validated using in vitro experiments. A renal tubular epithelial cell line (HK-2 cells) was exposed to hypoxia and reoxygenation (H/R) using air-tight conditions for five hours and treated with different concentrations of SE (25%, 50%, and 75%) to assess cell viability and apoptosis and determine the optimal experimental dose. Subsequently, H/R-injured HK-2 cells were pretreated with the optimal SE dose and then randomly divided into three groups, the SE, SE-SP600125 (inhibitor of JNK), and SE-NAC (antioxidant) groups. The cell vitality, apoptosis, and death were evaluated using the cell counting kit 8 (CCK8) and carboxyfluorescein succinimidyl ester/propidium iodide (CFSF/PI) staining. The apoptosis-related protein JNK and Caspase-3 were assessed by Western blot. Expression of JNK and Caspase-3 genes was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR).Results123 active components and 226 targets were identified from four herbs that composed the herb-compound-target network based on transcriptomics and network pharmacology analyses. The KEGG pathway analyses revealed that the mitochondrial apoptosis pathway was involved in the therapeutic AKI effects of SE. Cell vitality of H/R-induced HK-2 cells was obviously increased when treating them with SE, and the apoptosis was significantly inhibited, especially in the SE (50%) group at 4 and 12 h after modeling. Pretreatment with antioxidant NAC obviously prevented cell death compared to the SE (50%) group, while no obvious reduction of apoptosis was observed in the SP600125 group. JNK expression level was significantly increased in the SE (50%) group compared to the SP600125 (P < 0.01) and the NAC group (P < 0.05). Caspase-3 was downregulated in the SE (50%) group compared to the SP600125 (P < 0.01) and NAC group (P < 0.05). Caspase-3 activation in the SP600125 group was higher than that in the NAC group (P < 0.05). Moreover, the oxidative damage-dependent JNK/Caspase-3 pathway was identified in the H/R-injured HK-2 cells by inhibiting the JNK activation and oxidative damage.ConclusionsOur findings suggested that the H/R-triggered apoptosis in HK-2 cells was abrogated by SE by upregulating the oxidative damage-dependent JNK to trigger suppression of Caspase-3.

Project description:ObjectiveRenal ischemia-reperfusion (I/R) injury is a major cause of acute kidney injury (AKI) in transplanted kidneys. This study was aimed at exploring the role of PLK3 (polo-like kinase 3) in renal I/R injury, focusing on its relationship with oxidative stress-induced DNA damage and renal tubular epithelial cell (TEC) apoptosis.MethodsTRAP-seq data from the development dataset GSE52004 and the validation dataset GSE121191 were analyzed using GEO2R. PLK3 overexpression plasmids and targeted silencing siRNAs were used in a model of hypoxia/reoxygenation (H/R) injury, and rAAV-9-PLK3-KD were administered to C57BL/6J mice exposed to I/R injury. The ATM-specific inhibitor KU-60019 was used to block the DNA damage response (DDR). Western blotting was performed to measure DDR- and apoptosis-associated protein expression. Cell viability was measured by CCK-8 reagent, and apoptosis was examined by flow cytometry and TUNEL assay. Furthermore, the fluorescent probes H2DCFH-DA and DHE were used to measure ROS production in vitro. The MDA level and SOD activity were measured to assess oxidative stress in vivo. KIM-1 staining and Scr and BUN were used to evaluate kidney injury.ResultsThe mRNA and protein levels of PLK3 were markedly increased in the H/R injury and I/R injury models. GO terms showed that PLK3 was mainly involved in oxidative stress and DNA damage after renal I/R injury. Overexpression of PLK3 decreased cell viability and increased apoptosis. In contrast, targeted silencing of PLK3 expression decreased the Bax/Bcl-2 ratio by decreasing P53 phosphorylation, thereby reducing TEC apoptosis. Furthermore, KU-60019 reduced PLK3 activation and DDR-induced apoptosis, while overexpression of PLK3 reversed the mitigating effect of KU-60019 on TEC apoptosis. Similarly, rAAV-9-PLK3 KD mice exhibited a lower rate of TEC apoptosis and milder renal damage after I/R injury.ConclusionWe demonstrate for the first time that PLK3 is involved in oxidative stress-induced DNA damage and TEC apoptosis in renal I/R injury. Inhibition of PLK3 attenuates TEC apoptosis after I/R injury by blocking the ATM/P53-mediated DDR. Therefore, PLK3 may serve as a potential therapeutic target for ischemic AKI.

Dataset Information

Growth arrest and DNA damage 45? is required for caspase-dependent renal tubular cell apoptosis.

Publications

Growth arrest and DNA damage 45γ is required for caspase-dependent renal tubular cell apoptosis.

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