Abstract
BackgroundGrowth 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.MethodsPrimary 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.ResultsExpression 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.ConclusionsTo 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.
Highlights
Growth Arrest and DNA Damage 45γ (GADD45γ), an isoform of the GADD45 family of proteins, is a molecule which responses to environmental stresses by checking on the cell cycle [1], and by inducing apoptosis [2]
The activation of caspase-3 and caspase-7 as well as caspase-9 induced by cisplatin or cyclosporine A (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
GADD45γ in renal tubular cell apoptosis. This is the first study to show that GADD45γ is required for the caspasedependent apoptosis of renal tubular cells induced by nephrotoxic drugs
Summary
Growth Arrest and DNA Damage 45γ (GADD45γ), an isoform of the GADD45 family of proteins, is a molecule which responses to environmental stresses by checking on the cell cycle [1], and by inducing apoptosis [2]. Apoptosis is a critical mode of renal tubular cell death in acute kidney injury (AKI) and prevention of apoptosis was shown to protect renal function [3]. For our in vitro experiments, we selected the nephrotoxic drugs cisplatin and cyclosporine A (CsA) to evaluate the link between GADD45γ and renal tubular cell apoptosis. Nephrotoxicity by cisplatin involves necrosis as well as apoptosis of renal tubular cells, and the suppression of apoptosis has been shown to be protective against cisplatin-induced renal injury [10]. Apoptosis is a critical mode of renal tubular cell death in nephrotoxin-induced acute kidney injury. We investigated the role of GADD45γ in renal tubular cell apoptosis induced by nephrotoxic drugs.
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