Wogonin protects glomerular podocytes by targeting Bcl-2-mediated autophagy and apoptosis in diabetic kidney disease

Abstract

Diabetic kidney disease (DKD) is one of the microvascular complications of diabetes mellitus and a major cause of end-stage renal disease with limited treatment options. Wogonin is a flavonoid derived from the root of Scutellaria baicalensis Georgi, which has shown a potent renoprotective effect. But the mechanisms of action in DKD are not fully elucidated. In this study, we investigated the effects of wogonin on glomerular podocytes in DKD using mouse podocyte clone 5 (MPC5) cells and diabetic mice model. MPC5 cells were treated with high glucose (30 mM). We showed that wogonin (4, 8, 16 μM) dose-dependently alleviated high glucose (HG)-induced MPC5 cell damage, accompanied by increased expression of WT-1, nephrin, and podocin proteins, and decreased expression of TNF-α, MCP-1, IL-1β as well as phosphorylated p65. Furthermore, wogonin treatment significantly inhibited HG-induced apoptosis in MPC5 cells. Wogonin reversed HG-suppressed autophagy in MPC5 cells, evidenced by increased ATG7, LC3-II, and Beclin-1 protein, and decreased p62 protein. We demonstrated that wogonin directly bound to Bcl-2 in MPC5 cells. In HG-treated MPC5 cells, knockdown of Bcl-2 abolished the beneficial effects of wogonin, whereas overexpression of Bcl-2 mimicked the protective effects of wogonin. Interestingly, we found that the expression of Bcl-2 was significantly decreased in biopsy renal tissue of diabetic nephropathy patients. In vivo experiments were conducted in STZ-induced diabetic mice, which were administered wogonin (10, 20, 40 mg · kg^-1 · d^-1, i.g.) every other day for 12 weeks. We showed that wogonin administration significantly alleviated albuminuria, histopathological lesions, and p65 NF-κB-mediated renal inflammatory response. Wogonin administration dose-dependently inhibited podocyte apoptosis and promoted podocyte autophagy in STZ-induced diabetic mice. This study for the first time demonstrates a novel action of wogonin in mitigating glomerulopathy and podocytes injury by regulating Bcl-2-mediated crosstalk between autophagy and apoptosis. Wogonin may be a potential therapeutic drug against DKD.

Keywords: Bcl-2; apoptosis; autophagy; diabetic kidney disease; podocytes; wogonin.

Fig. 1. Effect of wogonin on HG-treated MPC5 cells’ viability and inhibition on HG-induced inflammation.

a The molecular structural formula of wogonin. b MTT assay to determine the effect of wogonin on viability of MPC5 cells and HG-treated MPC5 cells. c Western blot of WT-1, nephrin, and podocin in MPC5 cells. d Real-time PCR analysis of TNF-α, MCP-1, and IL-1β expression in MPC5 cells. e Western blot analysis of phosphorylated p65 (p-p65) in MPC5 cells. Results represent mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs NC. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs HG. HG high glucose, MG 5.5 mM glucose plus 24.5 mM mannitol, NC normal control, Wog wogonin

Fig. 2. Wogonin promotes HG-induced autophagy disorders.

b Western blot analysis of beclin-1, ATG7, LC3, and p62 in MPC5 cells. b Representative transmission electron microscopy images of autophagosomes in MPC5 cells, the arrows indicate autophagosomes. Scale bar = 500 nm. Results represent mean ± SEM of three independent experiments. **P < 0.01, ***P < 0.001 vs NC. ^##P < 0.01, ^###P < 0.001 vs HG. HG high glucose, NC normal control, Wog wogonin

Fig. 3. Wogonin inhibits HG-induced apoptosis.

a Western blot analysis of cleaved caspase-3, Bax, and Bcl-2 in MPC5 cells. b Real-time PCR analysis of Bcl-2 in MPC5 cells. c Flow cytometry analysis of PI/Annexin V-stained MPC5 cells. d TUNEL assay for MPC5 cells. Scale bar = 50 μm. Results represent mean ± SEM of three independent experiments. ***P < 0.001 vs NC. ^##P < 0.01, ^###P < 0.001 vs HG. Abbreviations: HG high glucose, NC normal control, Wog wogonin

Fig. 4. Prediction of wogonin molecular targets.

a Profiling of the predicted protein targets of wogonin using Discovery Studio 2017 software. b Molecular docking of wogonin binding to Bcl-2 crystal structure. c Immunohistochemistry analysis of Bcl-2 in human kidney. Scale bar = 50 μm. Data represent the mean ± SEM for 6 humans. d CETSA analysis in MPC5 cells. e Immunoprecipitation assay. Results represent mean ± SEM of three independent experiments. ***P < 0.001 vs NC. HG high glucose, NC normal control, Wog wogonin

Fig. 5. Wogonin fails to reduce the HG-induced cell injury, inflammatory response and promotes autophagy in Bcl-2-silenced MPC5 cells.

a Real-time PCR analysis of Bcl-2 in MPC5 cells. b Western blot analysis of Bcl-2 in MPC5 cells. c Western blot analysis of WT-1 and nephrin in MPC5 cells. d Western blot analysis of cleaved caspase-3, Beclin-1, ATG7, and p62 in MPC5 cells. Results represent means ± SEM for three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs NC. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs HG. ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001 vs Bcl-2 EV group. HG high glucose, Wog wogonin, EV empty vector, KD Knockdown, NC normal control

Fig. 6. Bcl-2 overexpression and treatment with wogonin have similar cellular protective roles.

a Real-time PCR analysis of Bcl-2 in MPC5 cells. b Western blot analysis of Bcl-2 in MPC5 cells. c Western blot analysis of WT-1 and nephrin in MPC5 cells. d Western blot analysis of cleaved caspase-3, Beclin-1, ATG7, and p62 in MPC5 cells. Results represent means ± SEM for three independent experiments. **P < 0.01, ***P < 0.001 vs NC. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs HG. ^$$P < 0.01, ^$$$P < 0.001 vs Bcl-2-OE group. HG high glucose, Wog wogonin, EV empty vector, OE overexpression, NC normal control

Fig. 7. Physical and biochemical markers and histopathology of type 1 diabetic mice.

a Analysis of urine albumin excretion. b Fasting blood glucose in different groups. c Kidney/body weight. d serum BUN assay. e serum Cr assay. f Twenty-four hours urinary protein quantitation. g Histological observations of kidney sections stained with periodic acid–Schiff (PAS) from different groups treated with or without wogonin. Scale bar = 50 μm. h Representative transmission electron microscopy images of podocyte foot processes and glomerular basement membrane. Scale bar = 1 μm. Results represent mean ± SEM for 6–8 mice. ***P < 0.001 vs NC. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs STZ. NC normal control, Wog wogonin, STZ streptozotocin

Fig. 8. Wogonin attenuates glomerular podocytes injury in type 1 diabetic mice.

a Immunohistochemistry analysis of WT-1 in mice kidney. Scale bar = 50 μm. b Immunohistochemistry analysis of nephrin in mice kidney. Scale bar = 50 μm. c Western blot analysis of WT-1, nephrin, and podocin in mice kidney. Data represent the mean ± SEM for 6–8 mice. *P < 0.05, ***P < 0.001 vs NC. ^#P < 0.05, ^###P < 0.001 vs STZ. NC normal control, Wog wogonin, STZ streptozotocin

Fig. 9. Wogonin attenuates renal inflammation in type 1 diabetic mice.

a Real-time PCR analysis of inflammation (TNF-α, MCP-1, IL-1β) indices in mice kidney samples. b Western blot analysis of phosphorylated p65 (p-p65) in mice kidney tissues. c Immunohistochemistry analysis of TNF-α, MCP-1, IL-1β in mice kidney tissues. Scale bar = 50 μm. Data represent the mean ± SEM for 6–8 mice. ***P < 0.001 vs NC. ^##P < 0.01, ^###P < 0.001 vs STZ. NC normal control, Wog wogonin, STZ streptozotocin

Fig. 10. Wogonin attenuates renal autophagy disorder in type 1 diabetic mice.

a Immunohistochemistry analysis of p62 in human kidney tissues. Scale bar = 50 μm. Data represent the mean ± SEM for six humans. b Western blot analysis of Beclin-1, ATG7, p62, and LC3 in mice kidney tissues. c Representative transmission electron microscopy images of autophagosomes in mice kidney tissues, the arrows indicate autophagosomes. Scale bar = 500 nm. Data represent the mean ± SEM for 6–8 mice. ***P < 0.001 vs NC. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs STZ. NC normal control, Wog wogonin, STZ streptozotocin

Fig. 11. Wogonin attenuates cell death in type 1 diabetic mice.

a Western blot analysis of cleaved caspase-3, Bax, and Bcl-2 in mice kidney tissues. b TUNEL assay in mice kidney tissues. apoptotic cell, red; DAPI, blue. c Immunohistochemistry analysis of Bcl-2 in mice kidney tissues. Scale bar = 50 μm. Results represent mean ± SEM for 6–8 mice. ***P < 0.001 vs NC. ^##P < 0.01, ^###P < 0.001 vs STZ. Abbreviations: NC normal control, Wog wogonin, STZ streptozotocin