FOXO3a Protects against Kidney Injury in Type II Diabetic Nephropathy by Promoting Sirt6 Expression and Inhibiting Smad3 Acetylation

Abstract

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease. Although numerous reports have demonstrated a correlation between epithelial-mesenchymal transition (EMT) and renal fibrosis, how these processes lead to tubular dysfunction remains unclear. Here, we show that FOXO3a protects kidneys from injury in type II DN by increasing Sirt6 expression, which deacetylates Smad3 and inhibits its transcriptional activity. The results showed that progressive EMT in the kidneys from db/db mice is associated with Sirt6 downregulation and involved in tubular injury and dysfunction. The reduction of Sirt6 levels in db/db mice resulted in progressive kidney injury, indicating the protective role of Sirt6. Furthermore, Sirt6 was shown to directly bind to Smad3, a key downstream mediator of TGF-β, and could deacetylate it to inhibit its nuclear accumulation and transcriptional activity in HK2 cells. Besides, we demonstrate that FOXO3a activates Sirt6 expression by binding to its promoter. shRNA-induced FOXO3a knockdown in the kidneys of db/db mice exacerbated tubular injury and renal function loss. Mechanistically, FOXO3a protects against kidney injury in type II DN through the Sirt6/Smad3 axis. Thus, the pharmacological targeting of FOXO3a-mediated Sirt6/Smad3 signaling pathways may provide a novel strategy for treating type II DN.

Figure 1

The progression of EMT HG-induced HK2 cells is associated with reduced Sirt6 levels. (a, b) HK2 cells were cultured in the HG medium for 72 h. (a) mRNA expression of fibrotic genes and Sirt6 analyzed by qPCR. (b) Protein expression of fibrotic genes and Sirt6 assessed by western blot analysis. (c–f) HK2 cells were transfected with si-NC or si-Sirt6 to knock down Sirt6 or transfected with the vehicle or the Sirt6 overexpression vector to increase Sirt6 expression and cultured in the Mann medium or HG medium for 72 h. (c) mRNA expression of fibrotic genes and Sirt6 analyzed by qPCR. (d) Protein expression of fibrotic genes and Sirt6 assessed by western blot analysis. (e) mRNA expression of tubular damage genes analyzed by qPCR. (f) Protein expression of tubular damage genes assessed by western blot analysis. (g, h) Representative images of cell migration and graphs showing the wound area quantification in HK2 cells. After 48 h of transfection, wounds were generated using a 200 μl pipette tip and cultured for another 48 h. HK2 cells transfected with si-NC or si-Sirt6 were cultured in the Mann medium, and cells transfected with the vehicle or the Sirt6 overexpression vector were cultured in the HG medium. (i) Representative images of Transwell assay and graphs showing migrated cells. After 24 h of seeding, HK2 cells were transfected with si-NC or si-Sirt6 cultured in the Mann medium and transfected with the vehicle or the Sirt6 overexpression vector cultured in the HG medium. The data are presented as the means ± SD. n = 3 experiments in (a–i). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.01.

Figure 2

Reduced Sirt6 levels contribute to the exacerbation of kidney injury in db/db mice. (a, b) Protein and mRNA were extracted from the kidneys of 20-week-old db/db and db/+ mice. (a) Sirt6 mRNA expression analyzed by qPCR. (b) Sirt6 protein expression assessed by western blot analysis. (c–j) The db/db or db/+ mice used in all experiments were 20 weeks old and were infected with sh-NC or sh-Sirt6 lentiviruses in the kidneys at 18 weeks old to knock down Sirt6 expression in vivo. (c–e) Graphs showing ACR, BUN, and proteinuria as analyzed by the ELISA kit. (f) Representative images of PAS staining of kidneys from mice. Scale bar, 40 μm. (g) Scoring of tubular injury. Proximal and distal tubules are marked with red and black arrows, respectively. “^” represents the brush border of proximal tubules. “^∗” represents the cellular debris in the lumen of proximal tubules. “^∗∗” represents the disorder proximal tubular structure, respectively. (h) α-SMA, E-cadherin, Sirt6, and Collagen I mRNA expression analyzed by qPCR. (i) Protein expression of α-SMA, E-cadherin, Sirt6, and Collagen I assessed by western blot analysis. (j) IHC analysis of α-SMA, E-cadherin, and Sirt6 expression in kidney tissue. (k) KIM-1 and NGAL mRNA expression analyzed by qPCR. (l) Protein expression of KIM-1 and NGAL assessed by western blot analysis. The data are presented as the means ± SD. n = 6 experiments in (a–e) and (h–l). n = 60 experiments in (g). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.01.

Figure 3

Sirt6 inhibits Smad3 transcriptional activity by deacetylating it and suppressing its nuclear localization. (a, b) Exogenous and endogenous co-IP assays performed to assess the binding and between Sirt6 and Smad3 and Smad3 acetylation in HK2 cells. (a) HK2 cells cultured in the Mann or HG medium were transfected with Flag-Smad3 or Flag-IgG. Proteins immunoprecipitated with an anti-Flag antibody were analyzed using anti-acetylation, anti-Smad3, anti-Sirt6, and anti-Flag antibodies. (b) HK2 cells were cultured in the Mann or HG medium for 72 h. Proteins immunoprecipitated with an anti-Smad3 antibody were analyzed using anti-acetylation, anti-Sirt6, and anti-Smad3 antibodies. (c) Endogenous co-IP analysis to assess the binding and between Sirt6 and Smad3 and Smad3 acetylation in the kidneys of db/+ or db/db mice. Proteins immunoprecipitated with an anti-Smad3 antibody were analyzed using anti-acetylation, anti-Sirt6, and anti-Smad3 antibodies. Proteins from whole-cell lysates analyzed using anti-GAPDH antibodies. (d–f) The luciferase activity for the SBE-Luc reporter assay. (d) Wild-type HK2 cells and HK2-Smad3^−/− cells cultured in the Mann medium were cotransfected with SBE-Luc plasmids with the si-NC, si-Sirt6, vehicle, and Sirt6 overexpression vector for 72 h, respectively. (e) Wild-type HK2 cells were transfected with SBE-Luc plasmids for 72 h. Different concentrations of OSS_128167 were added into the Mann medium to inhibit Sirt6 deacetylation at 24 h after transfection. (f) HK2-Smad3^−/− cells stably overexpressing wild-type or mutant Smad3 with K333A and K378A mutations were medium-cotransfected with SBE-Luc plasmids with the si-NC, si-Sirt6, vehicle, and Sirt6 overexpression vector, respectively, and cultured in the Mann medium for 72 h. (g, h) Nuclear proteins were extracted from HK2 cells or the kidneys of db/db and db/+ mice. (g) Smad3 protein expression in HK2 cells transfected with the si-NC, si-Sirt6, vehicle, and Sirt6 overexpression plasmid, as assessed by western blot analysis. (h) Smad3 protein expression in the kidneys of db/db or db/+ mice injected with sh-NC or sh-Sirt6 lentivirus, as assessed by western blot analysis. (i) Representative IF images of HK2 cells staining with Smad3 and DAPI in different conditions. The data are presented as the means ± SD. n = 3 experiments in (a–g) and (i). n = 6 experiments in (h). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.01.

Figure 4

FOXO3a regulates the EMT process in HK2 cells by binding to the Sirt6 promoter and increasing its expression. (a, b) HK2 cells were cultured in the HG medium for 72 h. (a) FOXO3a and Sirt6 mRNA expression analyzed by qPCR. (b) FOXO3a and Sirt6 protein expression assessed by western blot analysis. (c–f) HK2 cells cultured in the Mann medium were transfected with si-Sirt6 (c, d) or si-FOXO3a (e, f) to knock down Sirt6 or FOXO3a, respectively, or were transfected with si-NC for 72 h. HG-induced HK2 cells were transfected with Sirt6 (c, d) or FOXO3a (e, f) overexpression vectors to increase Sirt6 or FOXO3a expression, respectively, or were transfected with the vehicle for 72 h. (c, e) FOXO3a and Sirt6 mRNA expression analyzed by qPCR. (d, f) FOXO3a and Sirt6 protein expression assessed by western blot analysis. (g) Representative images of cell migration and graphs showing the wound area quantification for HK2 cells. After 48 h of transfection, wounds were generated with a 200 μl pipette tip and cultured for another 48 h. HK2 cells transfected with si-NC or si-FOXO3a were cultured in the Mann medium, and cells transfected with the vehicle or the FOXO3a overexpression vector were cultured in the HG medium. (h) Representative images of Transwell assay and graphs showing migrated cells. After 24 h of seeding, HK2 cells were transfected with si-NC or si-FOXO3a cultured in the Mann medium and transfected with the vehicle or the FOXO3a overexpression vector cultured in the HG medium. (i) The luciferase activity of promoter dual-luciferase assays. The -2083 to -1859 region of the Sirt6 promoter was inserted into the pGL3 vector. HK2 cells cultured in the Mann medium were cotransfected with pGL3, pRL-TK, and si-FOXO3a or FOXO3a for 48 h. (j) The ChIP RT-qPCR assay results were used to assess the enrichment of FOXO3a in the Sirt6 promoter region in HK2 cells cultured in the Mann medium for 72 h. The data are presented as the means ± SD. n = 3 experiments in (a–j). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.01.

Figure 5

Downregulation of FOXO3a leads to the deterioration of kidney injury in db/db mice. (a, b) Protein and mRNA were extracted from the kidneys of 20-week-old db/db and db/+ mice. (a) FOXO3a mRNA expression analyzed by qPCR. (b) FOXO3a protein expression assessed by western blot analysis. (c–j) The db/db or db/+ mice used in all experiments were 20 weeks old and were infected with sh-NC or sh-FOXO3a lentiviruses in the kidneys at 18 weeks old to knock down FOXO3a expression in vivo. (c–e) Graphs showing ACR, BUN, and proteinuria as analyzed by the ELISA kit. (f) Representative images of PAS staining of kidneys from mice. Scale bar, 40 μm. (g) Scoring of tubular injury. Proximal and distal tubules are marked with red and black arrows, respectively. “^” represents the brush border of proximal tubules. “^∗” represents the cellular debris in the lumen of proximal tubules. “^∗∗” represents the disorder proximal tubular structure, respectively. (h) IHC analysis of α-SMA, E-cadherin, and FOXO3a expression in kidney tissue. (i) α-SMA, E-cadherin, FOXO3a, and Collagen I mRNA expression analyzed by qPCR. (j) Protein expression of α-SMA, E-cadherin, FOXO3a, and Collagen I assessed by western blot analysis. (k) KIM-1 and NGAL mRNA expression analyzed by qPCR. (l) Protein expression of KIM-1 and NGAL assessed by western blot analysis. The data are presented as the means ± SD. n = 6 experiments in (a–e) and (h–l). n = 60 experiments in (g). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.01.