Polysulfide-mediated sulfhydration of SIRT1 prevents diabetic nephropathy by suppressing phosphorylation and acetylation of p65 NF-κB and STAT3

Abstract

Diabetic kidney disease is known as a major cause of chronic kidney disease and end stage renal disease. Polysulfides, a class of chemical agents with a chain of sulfur atoms, are found to confer renal protective effects in acute kidney injury. However, whether a polysulfide donor, sodium tetrasulfide (Na₂S₄), confers protective effects against diabetic nephropathy remains unclear. Our results showed that Na₂S₄ treatment ameliorated renal dysfunctional and histological damage in diabetic kidneys through inhibiting the overproduction of inflammation cytokine and reactive oxygen species (ROS), as well as attenuating renal fibrosis and renal cell apoptosis. Additionally, the upregulated phosphorylation and acetylation levels of p65 nuclear factor κB (p65 NF-κB) and signal transducer and activator of transcription 3 (STAT3) in diabetic nephropathy were abrogated by Na₂S₄ in a sirtuin-1 (SIRT1)-dependent manner. In renal tubular epithelial cells, Na₂S₄ directly sulfhydrated SIRT1 at two conserved CXXC domains (Cys371/374; Cys395/398), then induced dephosphorylation and deacetylation of its targeted proteins including p65 NF-κB and STAT3, thereby reducing high glucose (HG)-caused oxidative stress, cell apoptosis, inflammation response and epithelial-to-mesenchymal transition (EMT) progression. Most importantly, inactivation of SIRT1 by a specific inhibitor EX-527, small interfering RNA (siRNA), a de-sulfhydration reagent dithiothreitol (DTT), or mutation of Cys371/374 and Cys395/398 sites at SIRT1 abolished the protective effects of Na₂S₄ on diabetic kidney insulting. These results reveal that polysulfides may attenuate diabetic renal lesions via inactivation of p65 NF-κB and STAT3 phosphorylation/acetylation through sulfhydrating SIRT1.

Keywords: Diabetic nephropathy; Hydrogen sulfide; Polysulfides; Reactive oxygen species; SIRT1.

Graphical abstract

Fig. 1

Effects of Na₂S₄ on cell viability and apoptosis in HK-2 cells. (A) HK-2 cells were pre-incubated with Na₂S₄ (0, 3, 10, 30 μM) for 30 min, and then challenged by or HG (30 mM) for 48 h. Results showed that Na₂S₄ dose-dependently prevented HG-induced HK-2 cell viability decline. (B) HK-2 cells were pre-incubated with Na₂S₄ (0, 3, 10, 30 μM) for 30 min, and then challenged by or HG (30 mM) for 48 h. Effects of various doses of Na₂S₄ (0, 3, 10, 30 μM) on HG-induced LDH release. (C) HK-2 cells were pre-incubated with Na₂S₄ (30 μM) for 30 min, and then challenged by or HG (30 mM) for 48 h. EdU staining showed that Na₂S₄ (30 μM) attenuated HG-induced inhibition of HK-2 cell viability. (D–G) Representative blot images and quantitative analysis of Bax, Bcl-2, cleaved-caspase3 (C-cas3), and cleaved PARP (C-PARP). (H) Effects of Na₂S₄ (30 μM) on HG-induced cell apoptosis determined by TUNEL assay. *P < 0.05 vs. NG. †P < 0.05 vs. 0 μM or Vehicle (Veh). Scale bar = 200 μm n = 4 to 6.

Fig. 2

Effects of Na₂S₄ on inflammation response and oxidative stress in HK-2 cells. HK-2 cells were pre-incubated with Na₂S₄ (30 μM) for 30 min, and then challenged by or HG (30 mM) for 48 h. (A) Representative blot images and quantitative analysis of TNF-α, IL-1β, VCAM-1 and COX-2 showing that Na₂S₄ (30 μM) mitigated HG-induced inflammation response. (B) Relative mRNA levels of TNF-α, IL-1β, VCAM-1 and COX-2. (C–F) Representative blot images and quantitative analysis of p22^phox, NOX2, p47^phox and membrane p47^phox showing that Na₂S₄ (30 μM) mitigated HG-induced oxidative stress. (G) Effects of Na₂S₄ (30 μM) on HG-induced cell ROS production determined by DHE fluorescence staining. Scale bar = 200 μm *P < 0.05 vs. NG. †P < 0.05 vs. Vehicle (Veh). n = 4 to 6.

Fig. 3

Effects of Na₂S₄ on EMT process in HK-2 cells. HK-2 cells were pre-incubated with Na₂S₄ (30 μM) for 30 min, and then challenged by or HG (30 mM) for 48 h. (A–C) Representative blots and quantitative analysis of TGF-β1, α-SMA, E-cadherin showing that Na₂S₄ (30 μM) mitigated HG-induced fibrosis in HK-2 cells. (D–F) Relative mRNA levels of TGF-β1, α-SMA, E-cadherin. (G) Immunofluorescence staining for E-cadherin or α-SMA. Scale bar = 200 μm *P < 0.05 vs. NG. †P < 0.05 vs. Vehicle (Veh). n = 4 to 6.

Fig. 4

Effects of Na₂S₄ on SIRT1, phosphorylation and acetylation of p65 NF-κB and STAT3. (A) Effects of Na₂S₄ (30 μM) on SIRT1 protein levels. (B) Effects of HG (30 mM) on SIRT1 protein levels. (C) HK-2 cells were pre-incubated with Na₂S₄ (30 μM) for 30 min, and then challenged by or HG (30 mM) for 48 h. Pretreatment with Na₂S₄ (30 μM) reversed HG-triggered downregulation of SIRT1 protein expressions. (D–G) Effects of Na₂S₄ (30 μM) on the phosphorylation and acetylation levels of p65 NF-κB and STAT3. *P < 0.05 vs. 0 h or NG. †P < 0.05 vs. Vehicle (Veh). n = 4 to 6.

Fig. 5

Effects of SIRT1 mutation on phosphorylation and acetylation of p65 NF-κB and STAT3 as well as HK-2 cell injury induced by HG. HK-2 cells were transfected with wild type (WT) or mutated SIRT1 plasmids for 24 h before incubation of Na₂S₄ (30 μM) for 30 min, and were stimulated with HG for another 48 h. (A–D) SIRT1 mutation attenuated the suppressive effect of Na₂S₄ on HG-amplified p65 NF-κB/STAT3 phosphorylation and acetylation. (E) Representative blots and quantitative analysis of cleaved PARP (C-PARP), COX-2, p47^phox, and TGF-β1 showing that Na₂S₄ failed to attenuate HG-induced apoptosis, inflammation, oxidative stress, and fibrosis in HK-2 cells with SIRT1 mutation. *P < 0.05 vs. NG. †P < 0.05 vs. HG. ‡P < 0.05 vs. HG + Na₂S₄. #P < 0.05 vs. HG + Na₂S₄+M1 or HG + Na₂S₄+M2. n = 4 to 6.

Fig. 6

Na₂S₄ alleviated renal function and renal fibrosis in diabetic mice. (A) Body weight. (B) Fasting blood glucose. (C) BUN level. (D) Serum creatinine level. (E) Quantitative assessment of tubular injury. (F) Representative images of PAS staining. (G) Representative images of Sirius red staining. (H–J) Relative mRNA levels of collagen I, collagen III, CTGF, E-cadherin and α-SMA. (K–M) Representative blot images and quantitative analysis of TGF-β1, α-SMA and E-cadherin. Scale bar = 50 μm *P < 0.05 vs. Control. †P < 0.05 vs. Diabetes. The results were derived from 4 to 8 independent experiments. n = 4 to 8. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Na₂S₄ alleviated renal inflammation response in diabetic mice. (A) Representative blot images and quantitative analysis of TNF-α, IL-1β, VCAM-1 and COX-2. (B) Relative mRNA levels of TNF-α, IL-1β, VCAM-1 and COX-2. (C) The protein levels of TNF-α, IL-1β, and VCAM-1 measured by ELISA. (D) F4/80-positive macrophages in kidney tissues. Scale bar = 50 μm *P < 0.05 vs. Control. †P < 0.05 vs. Diabetes. n = 4 to 6.

Fig. 8

Na₂S₄ restrained renal oxidative stress in diabetic mice. (A–D) Representative blot images and quantitative analysis of p22^phox, NOX2, p47^phox and membrane p47^phox. (E) MDA content. (F) NADPH oxidase activity. (G) SOD activity. (H) GSH-Px activity. (I,L) DHE staining in kidney tissues. (J,M) DCFH-DA staining in kidney tissues. (K) Nitrotyrosine immunofluorescence in kidney tissues. Scale bar = 100 μm *P < 0.05 vs. Control. †P < 0.05 vs. Diabetes. n = 4 to 6.

Fig. 9

Na₂S₄ relieved renal cell apoptosis in diabetic mice. (A) Representative blots and quantitative analysis of Bax, Bcl-2, cleaved caspase-3 (c-cas3) and cleaved-PARP (c-PARP). (B) Representative images of TUNEL staining of kidney tissues. Scale bar = 100 μm *P < 0.05 vs. Control. †P < 0.05 vs. Diabetes. n = 4 to 6.

Fig. 10

Na₂S₄ upregulated SIRT1 to affect p65 NF-κB/STAT3 phosphorylation and acetylation in diabetic nephropathy. (A–B) Effects of Na₂S₄ on the phosphorylation and acetylation levels of p65 NF-κB. (C–D) Effects of Na₂S₄ on the phosphorylation and acetylation levels of STAT3. (E) Treatment with Na₂S₄ reversed diabetes-downregulated SIRT1 protein levels in the kidneys. *P < 0.05 vs. Control. †P < 0.05 vs. Diabetes. n = 4 to 6.