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. 2013 Dec:65:849-858.
doi: 10.1016/j.freeradbiomed.2013.08.169. Epub 2013 Aug 22.

Redox-sensitive glycogen synthase kinase 3β-directed control of mitochondrial permeability transition: rheostatic regulation of acute kidney injury

Zhen Wang  1 , Yan Ge  2 , Hui Bao  1 , Lance Dworkin  2 , Ai Peng  3 , Rujun Gong  4
Affiliations

Redox-sensitive glycogen synthase kinase 3β-directed control of mitochondrial permeability transition: rheostatic regulation of acute kidney injury

Zhen Wang et al. Free Radic Biol Med. 2013 Dec.

Abstract

Mitochondrial dysfunction plays a pivotal role in necroapoptotic cell death and in the development of acute kidney injury (AKI). Evidence suggests that glycogen synthase kinase (GSK) 3β resides at the nexus of multiple signaling pathways implicated in the regulation of mitochondrial permeability transition (MPT). In cultured renal tubular epithelial cells, a discrete pool of GSK3β was detected in mitochondria. Coimmunoprecipitation assay confirmed that GSK3β physically interacts with cyclophilin F and voltage-dependent anion channel (VDAC), key MPT regulators that possess multiple GSK3β phosphorylation consensus motifs, suggesting that GSK3β has a direct control of MPT. Upon a strong burst of reactive oxygen species elicited by the pro-oxidant herbicide paraquat, the activity of the redox-sensitive GSK3β was drastically enhanced. This was accompanied by augmented phosphorylation of cyclophilin F and VDAC, associated with MPT and cell death. Inhibition of GSK3β by either the selective inhibitor 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) or forced expression of a kinase-dead mutant obliterated paraquat-induced phosphorylation of cyclophilin F and VDAC, prevented MPT, and improved cellular viability. Conversely, ectopic expression of a constitutively active GSK3β amplified the effect of paraquat on cyclophilin F and VDAC phosphorylation and sensitized cells to paraquat-induced MPT and death. In vivo, paraquat injection elicited marked oxidant stress in the kidney and resulted in acute kidney dysfunction and massive tubular apoptosis and necrosis. Consistent with in vitro findings, the activity of GSK3β was augmented in the kidney after paraquat injury, associated with increased phosphorylation of cyclophilin F and VDAC and sensitized MPT. TDZD-8 blocked GSK3β activity in the kidney, intercepted cyclophilin F and VDAC phosphorylation, prevented MPT, attenuated tubular cell death, and ameliorated paraquat-induced AKI. Our data suggest that the redox-sensitive GSK3β regulates renal tubular injury in AKI by controlling the activity of MPT regulators.

Keywords: Acute kidney injury; Cyclophilin F; Free radicals; Glycogen synthase kinase 3β; Mitochondrial permeability transition; Paraquat; Voltage-dependent anion channel.

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Figures

Figure 1
Figure 1. GSK3β inhibition improves mitochondrial dysfunction following oxidative injury by paraquat in tubular cells
(a) The amount of reactive oxygen species as measured by the fluorescent DCF in cells treated with parquet (PQ, 0.25mM) for different periods of time (n = 6). (b) TKPT cells were exposed toTDZD-8 (10μM) and/or paraquat (0.25mM) for 24hours before the mitochondria were isolated. Mitochondria permeability transition was assessed by the decrease in spectrophotometric absorbance of calcium-challenged mitochondria at 540 nm. (c) TKPT cells were treated with TDZD-8 (10μM) and/or paraquat (0.25mM) for 24hours, and then the expression levels of GSK3β (in total cell lysates or mitochondrial fraction) and p-GSK3β (Ser9) (in total cell lysates) were determined by western blotting. Arbitrary units of p-GSK3β/GSK3β ratios expressed as immunoblot densitometric ratios of the molecules as folds of the control group. Abbreviation: Ctrl, Control; Mito, mitochondria. *P<0.05 versus other groups; (n=6).
Figure 2
Figure 2. GSK3β is a mitochondrial molecule and phosphorylates cyclophilin F and VDAC, key regulators of MPT
(a) Immunofluorescence staining of GSK3β and mitochondria (Mito) (by Mito Tracker Green) in TKPT cells. (b) Immunofluorescence staining of GSK3β and cyclophilin F (Cyp-F) in TKPT cells. (c) Immunofluorescence staining of GSK3β and VDAC in TKPT cells. (d) Lysates of TKPT cells were subjected to immunoprecipitation by an anti-cyclophilin F antibody, and immunoprecipitates were probed for cyclophilin F and GSK3β. (e) Lysates of TKPT cells were subjected to immunoprecipitation by an anti-VDAC antibody, and immunoprecipitates were probed for VDAC and GSK3β. (f) TKPT cells were transfected with indicated vectors. Relative kinase activity of GSK3β located in the cytosolic and mitochondrial fractions extracted from the transfected cells were estimated separately following treatment with paraquat (0.25mM) or vehicle for 24h. (g, h) Mitochondrial lysates were subjected to immunoprecipitation by an anti-cyclophilin F antibody or an anti-VDAC antibody, and immunoprecipitates were probed respectively for either cyclophilin F and p-serine (p-Ser), or VDAC and p-threonine (p-Thr). (i) Mitochondria permeability transition was assessed by the decrease in spectrophotometric absorbance of calcium-challenged mitochondria at 540 nm. (j) Cell viability of transfected cells was estimated by MTT assay. Abbreviation: DAPI, 4′, 6-diamidino-2-phenylindole; EV, empty vector; S9A, vectors encoding the constitutively active mutant GSK3β (S9A-GSK3β-HA/pcDNA3); KD, vectors encoding kinase-dead mutant GSK3β (GSK-3β-KD/pcDNA3). *P<0.05 vs other groups.
Figure 3
Figure 3. GSK3β inhibition improves general conditions and acute kidney dysfunction in paraquat-injured mice
Mice were subjected to paraquat (30 mg/kg) and/or TDZD-8 (10mg/kg) treatments. All mice were sacrificed 72 h after paraquat injury. (a) The kidney-to-body weight ratio was measured as the weight of two kidneys per body weight (mg/g). (b) Mice treated with TDZD-8 significantly reduced serum creatinine levels elicited by parauqat injury.(c) Blood urea nitrogen (BUN) was measured in mice on day 0, day 1, day 2 and day 3. TDZD-8 significantly reduced BUN levels elicited by parauqat injury. (d) Urine NGAL was measured by ELISA on urine samples collected from mice on day 3. *P<0.05 versus other groups (n=6).
Figure 4
Figure 4. Inhibition of GSK3β ameliorates histological injury and prevents tubular cell death in paraquat injured mice
Mice were subjected to paraquat (30 mg/kg) and/or TDZD-8 (10mg/kg) treatments. All mice were sacrificed 72h after paraquat administration. (a) Representative micrographs of hematoxylin eosin staining (× 200 and hgih-power view). (b) Histological changes were semi-quantitatively scored. (c) Western immunoblot analysis of NGAL expression in kidney homogenates. (d) Representative micrographs of TUNEL staining (counterstained with Evan’s blue; × 100). (e) TUNEL positive cells were counted and expressed as cells per mm2. (f) Western immunoblot analysis of kidney homogenates for pro-caspase-3 and active caspase-3 in mice. β-actin served as a loading control. Arbitrary units of active caspase-3/ pro-caspase-3 ratios expressed as immunoblot densitometric ratios of the molecules as folds of the control group. *P<0.05 versus other groups (n=6).
Figure 5
Figure 5. Inhibition of GSK3β obliterates phosphorylation of cyclophilin F and VDAC, desensitizes MPT and reduces oxidative stress in paraquat injured kidney
(a) Western immunoblot analysis of renal cytosol and mitochondria for p-GSK3β and GSK3β. (b) Kidney homogenates were subjected to immunoprecipitation by an anti-cyclophilin F antibody, and immunoprecipitates were probed for cyclophilin F or p-serine. (c) Kidney homogenates were subjected to immunoprecipitation by an anti-VDAC antibody, and immunoprecipitates were probed for VDAC or p-threonine. (d) Mitochondria were isolated from kidneys. Mitochondria permeability transition was assessed by the decrease in spectrophotometric absorbance of calcium-challenged mitochondria at 540 nm. (e) Representative micrographs of DCF staining of fresh kidney cryostat sections; (f) ROS generation was evaluated as the fluorescence intensity of DCF in the kidney tissues expressed as fold induction over the control group. *P<0.05 versus other groups (n=6).
Figure 6
Figure 6. Inhibition of GSK3β ameliorates liver injury in paraquat injured mice
Mice were subjected to paraquat (30 mg/kg) and/or TDZD-8 (10mg/kg) treatments. All mice were sacrificed 72h after paraquat injury. (a) Representative micrographs of TUNEL staining (counterstained with Evan’s blue; × 100). (b) TUNEL positive cells were counted and expressed as cells per mm2. (c) Western immunoblot analysis of active caspase-3 expression in kidney homogenates, β-actin was used as a loading control. Liver homogenates were subjected to immunoprecipitation by an anti-cyclophilin F or an anti-VDAC antibody, and immunoprecipitates were probed for either cyclophilin F and p-serine, or VDAC and p-threonine. *P<0.05 versus other groups (n=6).
Figure 7
Figure 7. Schematic diagram depicts the mechanisms of action of the GSK3β controlled MPT and the ensuing oxidative injury and cell death induced by paraquat
Paraquat induces reactive oxygen species (ROS) overproduction in renal tubular cells, followed by enhanced activity of GSK3β. Cyclophilin F and VDAC, key regulators of MPT, physically interact with GSK3β and are cognate substrates for GSK3β. Enhanced GSK3β activity promotes cyclophilin F and VDAC phosphorylation. Subsequently, increased activities of cyclophilin F and VDAC potentiate MPT pore opening upon ROS challenge, and this eventually aggravates necroapoptotic cell death, amplifies ROS release and results in acute kidney injury. TDZD-8, a highly selective small molecule inhibitor of GSK3β, blocked GSK3βactivity, diminishes cyclophilin F and VDAC phosphorylation, desensitizes MPT and thereby reduces oxidative injury and ameliorates necroapoptotic cell death and acute kidney injury. Abbreviation:ROS, reactive oxygen species; Cyp-F, cyclophilin F.
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