Alpha-lipoic acid ameliorates the epithelial mesenchymal transition induced by unilateral ureteral obstruction in mice

Abstract

The epithelial-to-mesenchymal transition (EMT) is one of mechanisms that induce renal interstitial fibrosis. Understanding EMT in renal fibrosis has important therapeutic implications for patients with kidney disease. Alpha-lipoic acid (ALA) is a natural compound with antioxidant properties. Studies for ALA are performed in acute kidney injury with renal tubular apoptosis, renal inflammation, and oxidative stress. We investigated the effects of ALA on EMT-mediated renal interstitial fibrosis in mice with unilateral ureteral obstruction (UUO). UUO mice developed severe tubular atrophy and tubulointerstitial fibrosis, with a robust EMT response and ECM deposition after 7 postoperative days. In contrast, ALA-treated UUO mice showed only moderate injury and minimal fibrosis and also larger reductions in the expression of ECM proteins, inflammatory factors, and EMT markers. ALA was shown to be involved in the suppression of infiltrating macrophages associated with EMT and the progression of interstitial fibrosis. It also lessened the destruction of the tubular basement membrane, by reducing the expression of matrix metalloproteinases. This is the first study to show that ALA modulates EMT in a UUO mouse model. Our results suggest that ALA merits further exploration as a therapeutic agent in the prevention and treatment of chronic kidney disease.

Figure 1. HE staining was performed to pathological changes at 7 days following UUO.

Semiquantitative analysis showed result of tubulointerstitial injury in the kidneys. The scoring injury was described in the “Materials and Methods”. ALA; only ALA treated group, ALA + Sham; ALA treated and no ureteral ligated group, UUO; no ALA treated, but ureteral ligated group, UUO + ALA; ALA treated and ureteral ligated group. Values are expressed as means ± SE (*P < 0.05). Scale bar, 50 μm.

Figure 2. ALA ameliorates renal fibrosis.

Masson’s trichrome staining (A) and immunohistochemical staining against collagen I (B) were performed. The severity of interstitial fibrosis and densitometric quantification was applied for collagen I positive areas were examined by densitometric quantification. ALA; only ALA treated group, ALA + Sham; ALA treated and no ureteral ligated group, UUO; no ALA treated, but ureteral ligated group, UUO + ALA; ALA treated and ureteral ligated group. Values are expressed as means ± SE (*P < 0.05). Scale bar, 50 μm. UUO1, enlarged image from insert in UUO. Arrow, tubular interstitial area. Asterisk, the dilated tubules.

Figure 3. ALA decreases EMT by UUO.

Immunoblot analysis was performed with a specific antibody against E-cadherin and α-SMA (A). Whole kidney were prepared and processed for immunoblot. β-actin was used as loading control and data were normalized against the density of β-actin by TotalLab TL100 v2006 software. Immunohistochemical staining was performed with a specific antibody against α-SMA (B). Scale bar, 50 μm. ALA; only ALA treated group, ALA + Sham; ALA treated and no ureteral ligated group, UUO; no ALA treated, but ureteral ligated group, UUO + ALA; ALA treated and ureteral ligated group. Values are expressed as means ± SE (*P < 0.05). Arrow, tubular interstitial area. Asterisk, the dilated tubules.

Figure 4. ALA ameliorates expression of TGF-β1 and pSmad.

Immunoblot analysis was performed with a specific antibody against TGF-β1 (A). β-actin was used as loading control and data were normalized against the density of β-action by TotalLab TL100 v2006 software. Immunohistochemical staining was performed with a specific antibody against TGF-β1 (B) and pSmad (C). The TGF-β1 expression was predominantly in the interstitium of UUO kidneys (arrow). Densitometric quantification for TGF-β1 and pSmad was applied to each group. Scale bar; 50 μm. ALA; only ALA treated group, ALA + Sham; ALA treated and no ureteral ligated group, UUO; no ALA treated, but ureteral ligated group, UUO + ALA; ALA treated and ureteral ligated group. Values are expressed as means ± SE (*P < 0.05). UUO1, enlarged image from insert in UUO. Arrow, tubular interstitial area. Asterisk, the dilated tubules.

Figure 5. ALA reduces renal fibrosis by regulating inflammation.

Immunoblot analysis was performed with a specific antibody against phosphorylated NF-κB and phosphorylated IκB-α. Data were normalized against the density of β-action by TotalLab TL100 v2006 software (A). Immunohistochemical staining was performed with a specific antibody against F4/80. A few F4/80-positive cells were observed in the peritubular areas (arrow) and interstitial foci (arrowhead) of ALA and ALA + sham kidneys (B) and MCP-1 (C). Scale bar; 100 μm. ALA; only ALA treated group, ALA + Sham; ALA treated and no ureteral ligated group, UUO; no ALA treated, but ureteral ligated group, UUO + ALA; ALA treated and ureteral ligated group. Values are expressed as means ± SE (*P < 0.05).

Figure 6. ALA decreases the expression of MMP-2 and MMP-9.

Immunohistochemical staining was performed with a specific antibody against MMP-2 (A) and MMP-9 (B). Densitometric quantification for MMP-2 and MMP-9 was applied to each group. ALA; only ALA treated group, ALA + Sham; ALA treated and no ureteral ligated group, UUO; no ALA treated, but ureteral ligated group, UUO + ALA; ALA treated and ureteral ligated group. Values are expressed as means ± SE (*P < 0.05). Scale bar; 50 μm. Asterisk, the dilated tubules. UUO1, enlarged image from insert in UUO. Arrow, tubular interstitial area. Asterisk, the dilated tubules.