Nicotine augments glomerular injury in a rat model of acute nephritis

Abstract

Background/aims: Epidemiologic studies suggest that cigarette smoke worsens the progression of renal injury in patients with glomerular diseases. The mechanisms involved have not been elucidated. These studies were designed to determine whether nicotine worsens markers of inflammation including glomerular cell proliferation and fibronectin deposition in an in vivo model of glomerular injury.

Methods: Sprague-Dawley rats were injected with anti-Thy1 antibody and given either tap water or nicotine in the drinking water until sacrifice at day 14. Fibronectin expression was measured by Western blot and immunohistochemistry. COX-2 expression was also determined by Western blot in the kidney cortex of rats treated with nicotine and in cultured human mesangial cells treated with nicotine.

Results: Anti-Thy1 antibody administration resulted in a significant increase in the number of cells per glomerulus that was further increased by the administration of nicotine. In nephritic rats, the administration of nicotine significantly increased fibronectin and COX-2 expression. In cultured human mesangial cells we also demonstrated that nicotine increases COX-2 expression and activity and that COX-2 mediates mesangial cell proliferation in response to nicotine.

Conclusion: Either in vivo or in vitro treatment with nicotine leads to activation of inflammatory mediators and hallmarks of glomerular injury, which may explain the mechanisms involved in the deleterious effects of cigarette smoking on renal disease.

Fig. 1

Nicotine increases glomerular cell number in rats with anti-Thy1 nephritis (n = 6). * p < 0.05 vs. tap water.

Fig. 2

Nicotine administration increases renal cortex fibronectin expression in rats with anti-Thy1 nephritis. A Representative Western blot (in duplicates) for fibronectin and α-actin used as control for unequal loading. B Densitometry analysis after reprobing the blot with an anti-α-actin antibody (n = 6). * p <0.05 vs. tap water; ^# p < 0.05 vs. vehicle rats.

Fig. 2

Nicotine administration increases renal cortex fibronectin expression in rats with anti-Thy1 nephritis. A Representative Western blot (in duplicates) for fibronectin and α-actin used as control for unequal loading. B Densitometry analysis after reprobing the blot with an anti-α-actin antibody (n = 6). * p <0.05 vs. tap water; ^# p < 0.05 vs. vehicle rats.

Fig. 3

Nicotine increases fibronectin expression (arrows) in rats with anti-Thy1 nephritis as assessed by immunohistochemistry. A Control. B Nicotine. C Anti-Thy1 alone. D Anti-Thy1 + nicotine.

Fig. 4

Nicotine increases renal cortex COX-2 expression in rats with anti-Thy1 nephritis. A Representative Western blot for COX-2 and actin used as control for unequal loading. Lane 1 = Anti-Thy1; lane 2 = anti-Thy1 + nicotine. B Densitometry data analysis after reprobing the blot with an anti-α-actin antibody (n = 6). * p < 0.05 vs. tap water.

Fig. 4

Nicotine increases renal cortex COX-2 expression in rats with anti-Thy1 nephritis. A Representative Western blot for COX-2 and actin used as control for unequal loading. Lane 1 = Anti-Thy1; lane 2 = anti-Thy1 + nicotine. B Densitometry data analysis after reprobing the blot with an anti-α-actin antibody (n = 6). * p < 0.05 vs. tap water.

Fig. 5

Nicotine increases COX-2 expression and activity in human mesangial cells. A Representative Western blot (in duplicates) for COX-2 and actin was used as control for unequal loading. B PGE₂ production by human mesangial cells exposed to nicotine (n = 3–6). C = Control; N = nicotine. * p < 0.05 vs. respective control.

Fig. 5

Nicotine increases COX-2 expression and activity in human mesangial cells. A Representative Western blot (in duplicates) for COX-2 and actin was used as control for unequal loading. B PGE₂ production by human mesangial cells exposed to nicotine (n = 3–6). C = Control; N = nicotine. * p < 0.05 vs. respective control.

Fig. 6

Nicotine-induced COX-2 expression in mesangial cells is prevented by the NADPH oxidase inhibitor DPI, and the ROS scavengers SOD and catalase. A Representative Western blot for fibronectin and actin used as control for unequal loading. Lane 1 = Control; lane 2 = nicotine; lane 3 = nicotine + DPI; lane 4 = nicotine + SOD; lane 5 = nicotine + catalase. B Densitometry data analysis performed after reprobing the blot with an α-actin antibody (n = 3–6). * p < 0.05 vs. control; ^# p < 0.05 vs. nicotine.

Fig. 6

Nicotine-induced COX-2 expression in mesangial cells is prevented by the NADPH oxidase inhibitor DPI, and the ROS scavengers SOD and catalase. A Representative Western blot for fibronectin and actin used as control for unequal loading. Lane 1 = Control; lane 2 = nicotine; lane 3 = nicotine + DPI; lane 4 = nicotine + SOD; lane 5 = nicotine + catalase. B Densitometry data analysis performed after reprobing the blot with an α-actin antibody (n = 3–6). * p < 0.05 vs. control; ^# p < 0.05 vs. nicotine.

Fig. 7

Nicotine-induced mesangial cell proliferation is prevented by the COX-2 inhibitors NS-398 and nimesulide (n = 3 in duplicate). * p < 0.05 vs. control; ^# p < 0.05 vs. nicotine.