Yonsei Med J.  2012 Nov;53(6):1165-1175. 10.3349/ymj.2012.53.6.1165.

Gamma Linolenic Acid Exerts Anti-Inflammatory and Anti-Fibrotic Effects in Diabetic Nephropathy

• Kim DH
• Yoo TH
• Lee SH
• Kang HY
• Nam BY
• Kwak SJ
• Kim JK
• Park JT
• Han SH
• Kang SW

• KMID: 1414260
• DOI: http://doi.org/10.3349/ymj.2012.53.6.1165

Abstract

PURPOSE
This study was undertaken to investigate the effects of gamma linolenic acid (GLA) on inflammation and extracellular matrix (ECM) synthesis in mesangial and tubular epithelial cells under diabetic conditions.
MATERIALS AND METHODS
Sprague-Dawley rats were intraperitoneally injected with either a diluent [n=16, control (C)] or streptozotocin [n=16, diabetes (DM)], and eight rats each from the control and diabetic groups were treated with evening primrose oil by gavage for three months. Rat mesangial cells and NRK-52E cells were exposed to medium containing 5.6 mM glucose and 30 mM glucose (HG), with or without GLA (10 or 100 microM). Intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), and fibronectin (FN) mRNA and protein expression levels were evaluated.
RESULTS
Twenty-four-hour urinary albumin excretion was significantly increased in DM compared to C rats, and GLA treatment significantly reduced albuminuria in DM rats. ICAM-1, MCP-1, FN mRNA and protein expression levels were significantly higher in DM than in C kidneys, and these increases were significantly abrogated by GLA treatment. In vitro, GLA significantly inhibited increases in MCP-1 mRNA expression and protein levels under high glucose conditions in HG-stimulated mesangial and tubular epithelial cells (p<0.05, respectively). ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1.
CONCLUSION
GLA attenuates not only inflammation by inhibiting enhanced MCP-1 and ICAM-1 expression, but also ECM accumulation in diabetic nephropathy.

Keyword

Gamma linolenic acid; experimental diabetic nephropathy; anti-inflammatory; anti-fibrotic

MeSH Terms

Animals
Anti-Inflammatory Agents/*therapeutic use
Blotting, Western
Chemokine CCL2/genetics/metabolism
Diabetic Nephropathies/*drug therapy/*metabolism
Enzyme-Linked Immunosorbent Assay
Fibronectins/genetics/metabolism
Intercellular Adhesion Molecule-1/genetics/metabolism
Rats
Rats, Sprague-Dawley
Real-Time Polymerase Chain Reaction
alpha-Linolenic Acid/*therapeutic use

Figure

• 

  Fig. 1 Renal MCP-1, ICAM-1, and fibronectin mRNA/18s rRNA ratios in control (C), C+gamma linolenic acid (GLA), diabetic (DM), and DM+GLA rats. There was a 2.1-fold increase in MCP-1 mRNA/18s rRNA, a 1.8-fold increase in ICAM-1 mRNA/18s rRNA, and a 2.7-fold increase in fibronectin mRNA/18s rRNA ratios in DM rats compared to C rats, and GLA treatment significantly abrogated these increases in mRNA/18s rRNA ratios in DM rats. *p<0.01 vs. C and C+GLA groups. †p<0.05 vs. DM group. MCP-1, monocyte chemoattractant protein-1; ICAM-1, intracellular adhesion molecule-1.

• 

  Fig. 2 Renal MCP-1 protein levels in C, C+GLA, DM, and DM+GLA rats. There was a 2.0-fold increase in renal MCP-1 protein levels in DM rats compared to C rats, and this increase in the DM rats was significantly ameliorated by GLA treatment. *p<0.01 vs. C and C+GLA groups. †p<0.05 vs. DM group. C, control; GLA, γ-linolenic acid; DM, diabetes; MCP-1, monocyte chemoattractant protein-1.

• 

  Fig. 3 Renal ICAM-1 and fibronectin protein expression in C, C+GLA, DM, and DM+GLA rats. There was a 3.2-fold increase in ICAM-1 and a 3.7-fold increase in fibronectin protein expression in DM rats compared to C rats, and these increases were significantly attenuated by administration of GLA. *p<0.01 vs. C and C+GLA groups. †p<0.05 vs. DM group. ICAM-1, intracellular adhesion molecule-1; C, control; GLA, γ-linolenic acid; DM, diabetes.

• 

  Fig. 4 Immunohistochemical staining for glomerular and tubulointerstitial ICAM-1, fibronectin, and ED-1 (as a marker of macrophage) in C, C+GLA, DM, and DM+GLA rats. Glomerular (A) and tubulointerstitial (B) ICAM-1 and fibronectin staining was significantly increased in DM rats compared to C rats, and GLA treatment significantly inhibited these increases in DM rats. The number of ED-1-positive cells was significantly higher in DM rats than in C rats, and GLA treatment significantly abrogated the number of glomerular and tubulointerstitial macrophages in DM rats. (C) IHC scores for ICAM-1 and fibronectin within the glomeruli and the tubulointerstitial area were significantly higher in DM rats relative to C rats, and GLA treatment significantly attenuated these increases in DM rats (×400). *p<0.05 vs. other groups. †p<0.01 vs. C and C+GLA groups. ‡p<0.05 vs. DM group. ICAM-1, intracellular adhesion molecule-1; C, control; GLA, γ-linolenic acid; DM, diabetes.

• 

  Fig. 5 MCP-1 mRNA/18s rRNA ratios and secreted MCP-1 protein levels in mesangial cells (A) and NRK-52E cells (B) exposed to 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), NG+10 or 100 µM GLA (NG+GLA), 30 mM glucose (HG), and HG+10 or 100 µM GLA (HG+GLA). There were 2.0- and 2.1-fold increases in MCP-1 mRNA/18s rRNA ratios in the HG-stimulated mesangial and NRK-52E cells, respectively, compared to the NG cells, and these increases in MCP-1 mRNA expression were significantly ameliorated by GLA treatment in a dose-dependent manner. There were 2.4- and 3.6-fold increases in MCP-1 levels in HG-stimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and GLA significantly attenuated these increases in MCP-1 levels in a dose-dependent manner. *p<0.01 vs. NG, NG+M, and NG+GLA groups. †p<0.05 vs. HG group. MCP-1, monocyte chemoattractant protein-1; GLA, γ-linolenic acid.

• 

  Fig. 6 ICAM-1 and fibronectin mRNA/18s rRNA ratios in mesangial cells (A) and NRK-52E cells (B) exposed to 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), NG+10 or 100 µM GLA (NG+GLA), 30 mM glucose (HG), and H+10 or 100 µM GLA (HG+GLA). There were 2.2- and 1.8-fold increases in ICAM-1 mRNA/18s rRNA ratios in HG-stimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and these increases in ICAM-1 mRNA/18s rRNA ratios were significantly abrogated by administration of GLA. There were 2.5-fold and 2.2-fold increases in fibronectin mRNA/18s rRNA ratios in HG-stimulated mesangial cells and NRK-52E cells, respectively, compared to NG cells, and GLA treatment significantly attenuated these increases in fibronectin mRNA/18s rRNA ratios in a dose-dependent manner. *p<0.01 vs. NG, NG+M, and NG+GLA groups. †p<0.05 vs. HG group. ICAM-1, intracellular adhesion molecule-1, GLA, γ-linolenic acid.

• 

  Fig. 7 Representative Western blots of ICAM-1 and fibronectin in cultured mesangial cells (A) and NRK-52E cells (B). There were significant increases in ICAM-1 and fibronectin protein expression in HG-stimulated cells as compared to NG cells, and these increases were significantly ameliorated with GLA treatment. *p<0.01 vs. NG, NG+M, and NG+GLA groups. †p<0.05 vs. HG group. ICAM-1, intracellular adhesion molecule-1, GLA, γ-linolenic acid.

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