Date Published: February 22, 2019
Publisher: Public Library of Science
Author(s): Gyu-Tae Shin, Hwa Joung Lee, Ji Eun Park, Partha Mukhopadhyay.
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.
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 , and by inducing apoptosis . 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 . With regard to kidney damage, we previously showed that GADD45γ contributes to the progression of chronic kidney disease in a mouse model of chronic tubular injury  and human chronic glomerulonephritis . To date, however, no data exists with regard to the role of GADD45γ in AKI, prompting us to investigate its role in apoptosis of renal tubular cells. Tubular cell death in AKI resulting from direct renal insults such as renal ischemia [6, 7], sepsis , and nephrotoxins [9–13] was shown to proceed through apoptosis. 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. Cisplatin is a widely used chemotherapy drug, but its use is limited by its nephrotoxicity . 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 . CsA was the first approved calcineurin inhibitor and has been extensively used in kidney transplantation to prevent acute rejection. However, ironically, CsA causes kidney injury [15, 16], and nephropathy caused by CsA has been associated with a marked increase in apoptosis of tubular and interstitial cells .
In this study, we sought to determine the role of GADD45γ in the death pathways of human renal tubular cells induced by the nephrotoxic drugs cisplatin and CsA which are known to cause renal tubular cell injury and related apoptosis. We found that the expression of GADD45γ gene was upregulated upon treatment with cisplatin and CsA, and that the inhibition of GADD45γ significantly reduced the apoptotic as well as necrotic cell death induced by the drugs. Accordingly, the overexpression of GADD45γ significantly augmented both modes of cell death induced by these drugs. The major executioner caspases, caspase-3 and caspase-7 and the initiator caspase, caspase-9 , were all activated by cisplatin and CsA, and such activation was significantly inhibited by GADD45γ knockdown, or significantly augmented by GADD45γ overexpression, indicating that the activation of caspases is dependent on the expression of GADD45γ. ZVAD-FMK, the pan-caspase inhibitor, effectively prevented apoptosis induced by these drugs, confirming the role of caspases in apoptotic cell death. The finding that necrosis (PI/Annexin V double stain) was more effectively prevented by ZVAD-FMK than the inhibitors of primary regulated necrosis, necrostatin-1 and ferrostatin-1, suggests that the observed necrosis should be a secondary event resulting from the progression of the apoptotic program towards necrosis . In addition, phospho-MLKL was not detected during the induction of cell death, indicating that necroptosis [18, 21, 22] was neither induced by cisplatin and CsA, nor affected by GADD45γ expression. Together, this set of evidence proves that GADD45γ is required for caspase activation and subsequent caspase-dependent apoptosis of renal tubular cell induced by nephrotoxic drugs.