BACKGROUND: Growth Arrest and DNA Damage 45gamma (GADD45gamma) 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 GADD45gamma 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 GADD45gamma expression was silenced, HRE cells were transduced with a plasmid encoding GADD45gamma-specific shRNA. The recombinant adenovirus containing the GADD45gamma gene was synthesized to overexpress GADD45gamma 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 GADD45gamma 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 GADD45gamma, and significantly augmented by the overexpression of GADD45gamma. The activation of caspase-3 and caspase-7 as well as caspase-9 induced by cisplatin or CsA was reduced by silencing of GADD45gamma, and was augmented by the overexpression of GADD45gamma, indicating that caspase activation is dependent on the expression of GADD45gamma. 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 GADD45gamma is required for the caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.
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