The Role of Akt2 in the Protective Effect of Fenofibrate against Diabetic Nephropathy

Abstract

Fenofibrate (FF) protects against diabetic nephropathy (DN) in type 1 diabetic (T1D) mice by upregulating the expression of fibroblast growth factor 21 (FGF21), leading to the activation of the Akt-mediated Nrf2 antioxidant pathways. Here, we examined which isoforms of Akt contribute to FF activation of FGF21-mediated renal protection by examining the phosphorylation and expression of three isoforms, Akt1, Akt2, and Akt3. T1D induced by a single intraperitoneal dose of streptozotocin (STZ) resulted in reduced phosphorylation of one isoform, Akt2, but FF treatment increased renal Akt2 phosphorylation in these and normal mice, suggesting a potential and specific role for renal Akt2 in FF protection against T1D. This was further confirmed using in vitro cultured HK-2 human kidney tubule cells exposed to high glucose (HG) with siRNA silencing of the Akt2 gene and STZ-induced diabetic Akt2-knockout mice with and without 3-month FF treatment. In normal HK-2 cells exposed to HG for 24 hours, FF completely prevented cell death, reduced total Akt expression and glycogen synthase kinase (GSK)-3β phosphorylation, increased nuclear accumulation of Fyn, and reduced nuclear Nrf2 levels. These positive effects of FF were partially abolished by silencing Akt2 expression. Similarly, FF abolished T1D-induced renal oxidative stress, inflammation, and renal dysfunction in wild-type mice, but was only partially effective in Akt2-KO mice. Furthermore, FF treatment stimulated phosphorylation of AMPKα, an important lipid metabolism mediator, which in parallel with Akt2 plays an important role in FF protection against HG-induced HK-2 cells oxidative stress and damage. These results suggest that FF protects against DN through FGF21 to activate both Akt2/GSK-3β/Fyn/Nrf2 antioxidants and the AMPK pathway. Therefore, FF could be repurposed for the prevention of DN in T1D patients.

Keywords: Akt2, fibroblast growth factor 21; diabetic nephropathy; nuclear factor erythroid 2-related factor 2; peroxisome proliferator-activated receptor α agonist..

Figure 1

Effects of the PPARα agonist fenofibrate (FF) on Akt isoforms in type 1 diabetic mice. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and then mice were administered FF (100 mg/kg) or vehicle by gavage every other day for 3 months. Western blotting was used to measure total and phosphorylated Akt1 (A, B), total and phosphorylated Akt2 (A, C), and total Akt3 (A, D) expression. Data are presented as the mean ± SD (n≥5). *, p < 0.05 vs. control mice (Ctrl); #, p < 0.05 vs. diabetic mice (DM).

Figure 2

Akt2 silencing increases high glucose (HG)-induced human renal tubular epithelial (HK-2) cell apoptosis and reduces the renal protective effect of FF. Human renal tubular epithelial (HK-2) cells were treated with either control or Akt2-specific siRNA, and then the cells were treated with combinations of HG and FF for 24 h. Western blotting, an Annexin V FITC/PI Apoptosis Detection Kit, and an Accuri C6 flow cytometer and Cell Quest Pro Software were used to assess the outcomes. (A) Total Akt2 expression. (B) Apoptosis. Q1 represents dead cells, Q2 represents non-viable apoptotic cells, Q3 represents normal cells, and Q4 represents viable apoptotic cells. Quantitative analysis is shown in (C). Data were collected from at least three independent experiments and are presented as the mean ± SD (n≥5). *, p < 0.05 vs. the corresponding Con-siRNA/Ctrl; #, p < 0.05 vs. the corresponding Con-siRNA/HG; &, p < 0.05 vs. the corresponding Con-siRNA/HG/FF; +, p < 0.05 vs. the corresponding Akt2-siRNA/Ctrl; §, p < 0.05 vs. the corresponding Akt2-siRNA/HG.

Figure 3

The role of Akt2 in the FF-induced protection of HK-2 cells against HG. Western blotting was used to measure Akt phosphorylation (A), and total and phosphorylated GSK-3β expression (B). Data were collected from at least three independent experiments and are presented as mean ± SD (n≥5). *, p < 0.05 vs. the corresponding Con-siRNA/Ctrl; #, p < 0.05 vs. the corresponding Con-siRNA/HG; &, p < 0.05 vs. the corresponding Con-siRNA/HG/FF; +, p < 0.05 vs. the corresponding Akt2-siRNA/Ctrl.

Figure 4

Downstream mediators of the FF-induced protection of HK-2 cells against HG. The expression of nuclear Fyn (AB) and Nrf2 (AC) was measured by western blotting. The transcriptional activity of Nrf2 was assessed by measuring the expression of its target genes, NQO-1 (D) and HO-1 (E), by qRT-PCR. (F) FGF21 expression. Data were collected from at least three independent experiments and are presented as mean ± SD (n≥5). *, p < 0.05 vs. the corresponding Con-siRNA/Ctrl; #, p < 0.05 vs. the corresponding Con-siRNA/HG; &, p < 0.05 vs. the corresponding Con-siRNA/HG/FF; +, p < 0.05 vs. the corresponding Akt2-siRNA/Ctrl; §, p < 0.05 vs. the corresponding Akt2-siRNA/HG.

Figure 5

Effects of FF on Akt2-KO and WT mice with diabetes. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and then mice were administered FF (100 mg/kg) or vehicle by gavage every other day for 3 months. Body mass (A), fasting glucose (B), and urinary albumin-to-creatinine ratio (UACR, C) were measured or calculated at the end of the treatment period. Renal histology (D) and glycogen deposition (purple) (E) were assessed following hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining of kidney sections (400 ×, scale bar 100 μm), respectively. Immunohistochemistry (IHC) for fibroblast growth factor 21 (FGF21) (F; brown: positive staining) was performed in kidney sections (400 ×, scale bar 100 μm), followed by quantitative analysis (G). Data are presented as the mean ± SD (n≥5). *, p < 0.05 vs. the corresponding WT/Ctrl; #, p < 0.05 vs. the corresponding WT/DM; &, p < 0.05 vs. the corresponding WT/DM/FF; +, p < 0.05 vs. the corresponding Akt2-KO/Ctrl; §, p < 0.05 vs. the corresponding Akt2-KO/DM.

Figure 6

Akt2 gene deletion partially abolishes the protective effect of FF against diabetes-associated renal damage. Renal oxidative damage was evaluated by western blotting for 4-hydroxy-2-nonenal (4-HNE) (AB). Renal inflammation was identified by western blotting for tumor necrosis factor-α (TNF-α) (AC). The severity of the renal fibrotic response was reflected in greater expression of connective tissue growth factor (CTGF), demonstrated using western blotting (AD). Masson's staining (E) was used to detect collagen fibers (blue) in kidney sections (400 ×, scale bar 100 μm). Semi-quantitative data generated from the Masson's-stained sections are presented in F. Data are presented as the mean ± SD (n≥5). *, p < 0.05 vs. the corresponding WT/Ctrl; #, p < 0.05 vs. the corresponding WT/DM; &, p < 0.05 vs. the corresponding WT/DM/FF; +, p < 0.05 vs. the corresponding Akt2-KO/Ctrl; §, p < 0.05 vs. the corresponding Akt2-KO/DM.

Figure 7

The role of AMPK in the FF-induced protection of HK-2 cells against HG. HK-2 cells were treated with either the control or Akt2-specific siRNA, and then the cells were treated with combinations of HG, FF and AMPK inhibitor (compound C, CC) for 24 h. Western blotting was used to measure total and phosphorylated AMPK expression (A). Renal oxidative damage was evaluated by western blotting for 4-HNE (BC, BD). Renal inflammation was detected by western blotting for TNF-α (BE, BF). Data were collected from at least three independent experiments and are presented as mean ± SD (n≥5). *, p < 0.05 vs. the corresponding Con-siRNA/Ctrl or Akt2-siRNA/Ctrl; #, p < 0.05 vs. the corresponding Con-siRNA/HG or Akt2-siRNA/HG; &, p < 0.05 vs. the corresponding Con-siRNA/HG/FF or Akt2-siRNA/HG/FF; +, p < 0.05 vs. the corresponding Con-siRNA+CC/Ctrl or Akt2-siRNA+CC/Ctrl; §, p < 0.05 vs. the corresponding Con-siRNA+CC/HG.

Figure 8

The mechanism of the protective effect of fenofibrate against diabetic nephropathy. Oxidative stress is a key pathogenic factor in the development of DN. Under diabetic conditions, glucose metabolism is impaired and excessive amounts of reactive oxygen species (ROS) are produced, which accumulate in the kidney and induce oxidative stress. Oxidative stress causes renal cellular injury and apoptosis, followed by inflammation and fibrosis, which ultimately leads to renal dysfunction. Our previous study showed that FF-induced renal protection from diabetes is mediated through an upregulation of FGF21, and this in turn stimulates Akt/GSK-3β/Fyn-mediated activation of the Nrf2 anti-oxidative pathway. The present study reveals that the protective effect of FF/FGF21 on DN is dependent on Akt2. The activation of the Akt2/GSK-3β/Fyn pathway increases the nuclear translocation of Nrf2 by inhibiting the nuclear accumulation of Fyn, and induces antioxidant gene expression. However, AMPK activation can also alleviate the metabolic defects, and both pathways prevent renal oxidative stress, inflammation, and remodeling.