Inhibition of glycogen synthase kinase-3β prevents NSAID-induced acute kidney injury

Abstract

Clinical use of nonsteroidal anti-inflammatory drugs (NSAIDs) like diclofenac (DCLF) is limited by multiple adverse effects, including renal toxicity leading to acute kidney injury. In mice with DCLF-induced nephrotoxicity, TDZD-8, a selective glycogen synthase kinase (GSK)3β inhibitor, improved acute kidney dysfunction and ameliorated tubular necrosis and apoptosis associated with induced cortical expression of cyclooxygenase-2 (COX-2) and prostaglandin E2. This renoprotective effect was blunted but still largely preserved in COX-2-null mice, suggesting that other GSK3β targets beyond COX-2 functioned in renal protection. Indeed, TDZD-8 diminished the mitochondrial permeability transition in DCLF-injured kidneys. In vitro, GSK3β inhibition reinstated viability and suppressed necrosis and apoptosis in DCLF-stimulated tubular epithelial cells. DCLF elicited oxidative stress, enhanced the activity of the redox-sensitive GSK3β, and promoted a mitochondrial permeability transition by interacting with cyclophilin D, a key component of the mitochondrial permeability transition pore. TDZD-8 blocked GSK3β activity and prevented GSK3β-mediated cyclophilin D phosphorylation and the ensuing mitochondrial permeability transition, concomitant with normalization of intracellular ATP. Conversely, ectopic expression of a constitutively active GSK3β abolished the effects of TDZD-8. Hence, inhibition of GSK3β ameliorates NSAID-induced acute kidney injury by induction of renal cortical COX-2 and direct inhibition of the mitochondrial permeability transition.

Figure 1. GSK3β inhibition protects mice from DCLF-induced renal tubular injury and cell apoptosis in vivo

Representative micrographs of hematoxylin and eosin (HE) staining (A, C, E, G, I) and TUNEL staining (B, D, F, H, J) of kidney specimens. A, B: Group Ctrl, control (vehicle treated); C, D: Group T, TDZD-8 (5 mg/kg); E, F: Group D, DCLF (200 mg/Kg); G, H: Group T-L+D, low dose TDZD-8 (1 mg/Kg)+DCLF (200 mg/Kg); I, J: Group T+D, TDZD-8 (5 mg/Kg)+DCLF (200 mg/Kg). K, L: Quantitative analysis of tubular injury and TUNEL-labeled cells among the groups. The results are expressed as the mean ± SD (n=6). *P<0.05, versus Group Ctrl; #P<0.05, versus Group D. DCLF elicited injury to both proximal and distal tubular cells. TUNEL-labeled nuclei were revealed as bright spots in the renal cortex in kidneys from DCLF-treated mice. TDZD-8 significantly attenuated both tubular necrosis and apoptosis in mouse kidneys.

Figure 2. Effects of TDZD-8 on GSK3β phosphorylaion and the levels of COX-2 and PGE₂ in kidney cortices of DCLF-treated mice

A: Western blot analysis of GSK3β and p-GSK3β in cortical homogenate. B: Western blot analysis of COX-1 and COX-2 in cortical homogenate. C: Analysis of cortical PGE₂ in cortical homogenate. Group Ctrl, control (vehicle treated); Group T, TDZD-8 (5 mg/kg); Group D, DCLF (200 mg/Kg); Group T-L+D, TDZD-8 (1 mg/Kg)+DCLF (200 mg/Kg); Group T+D, TDZD-8 (5 mg/Kg)+DCLF (200 mg/Kg). *P<0.05, versus Group Ctrl; #P<0.05, versus Group D. COX-1 expression was maintained and COX-2 expression was increased following treatment with DCLF. GSK3β inhibition led to a further increase in COX-2 expression in the renal cortices of DCLF-treated mice. Accordingly, PGE₂ synthesis was increased in TDZD-8 treated mice.

Figure 3. GSK3β inhibition protects COX-2 −/− mice from DCLF-induced renal tubular injury

A: mRNA expression of COX-2 detected by RT-PCR in the kidneys of wild-type (WT) and COX-2 knockout (KO) mice. B: Tubular injury score of WT and COX-2 KO mice treated with DCLF (200 mg/Kg) and/or TDZD-8 (5 mg/Kg). C: Serum creatinine (Scr) of WT and COX-2 KO mice treated with DCLF (200 mg/Kg) and/or TDZD-8 (5 mg/Kg). D: Representative micrographs of hematoxylin and eosin staining of kidney specimens from COX-2 KO mice treated with DCLF (200 mg/Kg) and/or TDZD-8 (5 mg/Kg). Ctrl, control (vehicle treated). *P<0.05, versus control; #P<0.05, versus DCLF-treated mice. DCLF produced injury to both proximal and distal tubular cells. This kidney protective effect of TDZD-8 was blunted but still largely preserved in COX-2 null mice.

Figure 4. Effects of GSK3β inhibition on the induction of the MPT by DCLF in mouse kidneys

A: Mitochondrial swelling was spectrophotometrically monitored at 540 nm following induction with 20 M CaCl₂ for 20 min. B: Change in absorbance at OD540 nm after 20 min. C: Correlation between decreased absorbance at 540 nm and the renal tubular injury score. D: Correlation between decreased absorbance at 540 nm and the number of apoptotic cells in renal tissues. Group Ctrl, control (vehicle treated); Group T, TDZD-8 (5 mg/kg); Group D, DCLF (200 mg/Kg); Group T-L+D, TDZD-8 (1 mg/Kg)+DCLF (200 mg/Kg); Group T+D, TDZD-8 (5 mg/Kg)+DCLF (200 mg/Kg). *P<0.05 versus Group Ctrl; #P<0.05 versus Group D. The decrease in absorbance at 540 nm was negatively correlated with the number of apoptotic cells and the renal tubular injury scores in mouse kidneys. Mitochondria that were isolated from kidneys treated with DCLF displayed an increased rate of mitochondrial swelling and a greater decrease in absorbance at 540 nm versus the controls, while TDZD-8 treatment ameliorated this process in a dose-dependent fashion.

Figure 5. Effects of GSK3β inhibition on the viability of renal tubular epithelial cells injured with DCLF

A: Viability of TKPT cells following treatment with different doses of DCLF (DCLF, 0, 100, 200, 400 μM) for 24 h. *P<0.05 versus normal controls; n=6. B: Survival rate of TKPT cells after treatment with 200 μM DCLF for different times. *P<0.05 versus the beginning; n=6. C: Survival rate of TKPT cells pretreated with different doses of TDZD-8 (0, 5, 10, 20 μM) and then treated with 200 μM DCLF for 24 h. *P<0.05 versus DCLF-treated cells; n=6. D: Survival rate of TKPT cells expressing either empty vector or S9A-GSK3β after treatment with 200 μM DCLF, 10 μM TDZD-8 or combined treatment. EV, empty vector transfected TKPT cells; S9A, S9A-GSK3β transfected TKPT cells; Ctrl, control; T, TDZD-8 10 μM; D, DCLF 200 μM. *P<0.05 versus control cells; #P<0.05 versus the value of empty vector-transfected cells treated with DCLF. Treatment with DCLF markedly decreased cell viability in a time- and dose-dependent manner, while treatment with TDZD-8 improved survival rates (P<0.05). In contrast, the supportive effect of TDZD-8 was blunted in cells transfected with vectors carrying S9A-GSK3β, a mutant of GSK3β that is unable to be phosphorylated at serine 9.

Figure 6. Effect of GSK3β inhibition on the apoptosis of TKPT cells treated with DCLF

A: TKPT cells were treated as indicated, fixed and exposed to TdT-mediated dUTP nick-end labeling (TUNEL) to assess apoptosis. B: Quantitative assessment of cell apoptosis. Cells in a ×200 field were manually counted in 10 fields per slide, with three replicates. EV, empty vector transfected TKPT cells; S9A, S9A-GSK3β atransfected TKPT cells; Ctrl, control; T, TDZD-8 10 M; D, DCLF 200 M. *P<0.05 versus control cells; #P<0.05 versus empty vector transfected cells treated with DCLF; n=6. There was a significant increase in the number of apoptotic cells following exposure to DCLF versus controls after 24 h (P<0.05). The number of apoptotic cells was decreased to approximately five per field in cultures pre-treated with TDZD-8, while the anti-apoptotic effect of TDZD-8 was attenuated in cells expressing S9A-GSK3β.

Figure 7. Effects of GSK3β inhibition on the necrosis of TKPT cells exposed to DCLF

A: Necrotic cell death was assessed using the propidium iodide exclusion assay in live cells. B: Quantitative assessment of cell necrosis by propidium iodide exclusion assay. Cells in a ×200 field were manually counted in 10 fields per slide, with three replicates. C: Necrotic cell death was assessed by lactate dehydrogenase (LDH) assay of the conditioned medium. EV, empty vector transfected TKPT cells; S9A, S9A-GSK3β transfected TKPT cells; Ctrl, control; T, TDZD-8 10 M; D, DCLF 200 M. *P<0.05 versus control cells; #P<0.05 versus empty vector transfected cells treated with DCLF; n=6. There was a significant increase in the number of necrotic cells and in LDH release in cultures exposed to DCLF versus controls after 24 h (P<0.05). TDZD-8 treatment decreased the number of necrotic cells and the LDH level, while the effects of TDZD-8 were attenuated in cells transfected with S9A-GSK3β.

Figure 8. Effects of GSK3β inhibition on MPT in TKPT cells exposed to DCLF

A: TMRM was used to detect the onset of the MPT by fluorescence microscopy. B: Mitochondrial swelling was spectrophotometrically monitored at 540 nm in mitochondria that had been induced with 20 M CaCl₂ for 20 min. EV, empty vector transfected TKPT cells; S9A, S9A-GSK3β transfected TKPT cells; Ctrl, control; T, TDZD-8 10 M; D, DCLF 200 M. *P<0.05 versus control cells; #P<0.05 versus empty vector transfected cells treated with DCLF; n=6. C: Correlation between the decrease in absorbance at 540 nm and the number of apoptotic or necrotic cells. Mitochondria that were isolated from cells treated with DCLF displayed quenching of TMRM and decreased absorbance at 540 nm compared to mitochondria from control cells. The extent of the decrease in absorbance at 540 nm was correlated with both the number of apoptotic and necrotic cells. Pretreatment with TDZD-8 inhibited the quenching of TMRM and restored the capacity of mitochondria that had been subjected to DCLF to accumulate calcium. All of these effects were blunted in cells expressing S9A-GSK3β.

Figure 9. Effect of GSK3β inhibition on intracellular ATP content in TKPT cells treated with DCLF

ATP concentration was measured using the luciferase method. EV, empty vector transfected cells; S9A, S9A-GSK3β transfected TKPT cells; Ctrl, control; T, TDZD-8 10 M; D, DCLF 200 M. *P<0.05 versus control cells; #P<0.05 versus empty vector transfected cells treated with DCLF; n=6. Treatment with DCLF significantly decreased ATP production. Treatment with TDZD-8 increased intracellular ATP, and the effect was blunted in cells transfected with S9A-GSK3β.

Figure 10. Effect of DCLF on redox-sensitive GSK3β in TKPT cells

TKPT cells were treated with 200 M DCLF or pretreated with 1 M rotenone (Rot) or 1 M stigmatellin (Stig). A: ROS formation was evaluated by the oxidation of DCFH-DA to the fluorescent DCF in TKPT cells. B: NADH dehydrogenase activity in mitochondrial extracts was compared by the fluorescence of NADH at the fifth minute after the start of assay. C: GSK3β activity was assayed by measuring the incorporation of ³²P into the substrate. D: Western blot analysis of GSK3β and p-Ser9-GSK3β expression in cells. *P<0.05 versus control cells; #P<0.05 versus cells treated with DCLF; n=6. DCLF treatment induced ROS production and GSK3β activation in TKPT cells but did not affect NADH dehydrogenase activity. Stigmatellin significantly suppressed ROS production and GSK3β activation in DCLF-treated TKPT cells, whereas rotenone had no effect.

Figure 11. GSK3β colocalizes with cyclophilin D, a component of the MPT pore complex, and regulates its activation

A: Representative images of the co-localization of GSK3β with mitochondria in TKPT cells. B: TKPT cell mitochondria were purified, immunoprecipitated with anti-cyclophilin D antibody, and immunoblotted with anti-GSK3β antibody. C: TKPT cell mitochondria were separated, immunoprecipitated with anti-cyclophilin D antibody, and immunoblotted with anti-phosphorylated-serine antibody. EV, empty vector transfected cells; S9A, S9A-GSK3β transfected TKPT cells; Ctrl, control; T, TDZD-8 10 M; D, DCLF 200 M. *P<0.05 versus control cells; #P<0.05 versus empty vector transfected cells treated with DCLF; n=6. GSK3β co-localized with cyclophilin D in the mitochondria. DCLF treatment significantly increased the phosphorylation of cyclophilin D. Inhibition of GSK3β by TDZD-8 suppressed the phosphorylation of cyclophilin D, whereas this effect was diminished in cells transfected with S9A-GSK3β.

Figure 12. GSK3β inhibition prevents NSAID-induced acute kidney injury by dual mechanisms

Over-inhibition of prostaglandin synthesis by NSAIDs leads to renal ischemia, a decline in glomerular hydraulic pressure and acute renal failure. DCLF is also able to injure renal tubular epithelial cells directly by triggering ROS production, enhancing the activity of redox-sensitive GSK3β, and thereby promoting cyclophilin D phosphorylation and the ensuing MPT pore opening. GSK3β inhibition prevents NSAID-induced acute kidney injury via both the induction of cortical cyclooxygenase-2 and the inhibition of the MPT.