Nicotine worsens the severity of nephropathy in diabetic mice: implications for the progression of kidney disease in smokers

Abstract

Epidemiological studies have established the role of cigarette smoking as a risk factor in the progression of chronic kidney disease, including diabetic nephropathy. We have previously reported that nicotine promotes mesangial cell proliferation and hypertrophy via activation of nonneuronal nicotinic acetylcholine receptors and that nicotine worsens renal injury in a model of acute glomerulonephritis (Jaimes E, Tian RX, Raij L. Am J Physiol Heart Circ Physiol 292: H76-H82, 2007; Jaimes EA, Tian RX, Joshi M, Raij L. Am J Nephrol 29: 319-326, 2009). These studies were designed to test the hypothesis that nicotine worsens renal injury in db/db mice, a well-established model of diabetic nephropathy, and that reactive oxygen species play an important as mediators of these effects. For these studies, nicotine (100 μg/ml) was administered in the drinking water to control and db/db mice for 10 wk. Blood pressure was measured by the tail-cuff method, and urine was collected for proteinuria. At death, kidneys were collected for histology and molecular biology. The administration of nicotine did not result in significant changes in blood pressure or blood glucose and resulted in cotinine levels similar to those found in the plasma of smokers. In diabetic mice, the administration of nicotine significantly increased urinary protein excretion (1-fold), glomerular hypertrophy, and mesangial area (∼20%). These changes were accompanied by significant increases in NADPH oxidase 4 (∼30%) and increased nitrotyrosine and Akt expression. In vitro, we determined that nicotine has additive effects to high glucose on reactive oxygen species generation and Akt phosphorylation in human mesangial cells. These findings unveil novel mechanisms that may result in the development of novel strategies in the treatment and prevention of diabetic nephropathy in smokers.

Fig. 1.

Effects of nicotine on glomerular hypertrophy and mesangial expansion in diabetic mice. A: diabetic mice had significant increases in total glomerular and mesangial areas by administration of nicotine in the drinking water. The administration of nicotine to nondiabetic mice resulted in a small and nonsignificant increase in glomerular and mesangial areas (n = 10/group). *P < 0.05 vs. control. #P < 0.05 vs. db/db mice on tap water. B: representative photomicrograph of periodic acid-Schiff-stained slides. A: control nondiabetic mice on tap water. B: nondiabetic mice on receiving nicotine in the drinking water. C: diabetic mice on tap water. D: diabetic mice receiving nicotine in the drinking water.

Fig. 2.

Effects of nicotine on urinary albumin excretion in diabetic mice. Diabetic mice had significant increases in urinary protein excretion compared with nondiabetic mice that was significantly increased by the administration of nicotine in the drinking water. In nondiabetic mice, the administration of nicotine did not modify urinary protein excretion (n = 10/group). *P < 0.05 vs. control. #P < 0.05 vs. db/db mice on tap water.

Fig. 3.

Effects of nicotine on cortical fibronectin expression in diabetic mice. Diabetic mice had significant increases in cortical expression of fibronectin, as assessed by Western blotting, that was significantly increased by the administration of nicotine in the drinking water. The administration of nicotine to nondiabetic mice resulted in a small and nonsignificant increase in fibronectin expression. A: representative Western blot for fibronectin and β-actin, which was used to control for loading. B: densitometric data analysis for cortical fibronectin expression (n = 10/group). OD, optical density. *P < 0.05 vs. control. #P < 0.05 vs. db/db mice.

Fig. 4.

Effects of nicotine on cortical NADPH oxidase (NOX) 4 expression in diabetic mice. Diabetic mice had significant increases in cortical expression of NOX4, as assessed by Western blotting, that was significantly increased by the administration of nicotine in the drinking water. The administration of nicotine to nondiabetic mice did not modify cortical NOX4. A: representative Western blot for NOX4 and β-actin, which was used to control for loading. B: densitometric data analysis for cortical NOX4 expression (n = 10/group). *P < 0.05 vs. control. #P < 0.05 vs. db/db mice.

Fig. 5.

Effects of nicotine on cortical nitrotyrosine expression in diabetic mice. Diabetic mice had significant increases in cortical expression of nitrotyrosine, as assessed by Western blotting, that was further increased by the administration of nicotine in the drinking water. A: representative Western blot for nitrotyrosine and tubulin, which was used to control for loading. B: densitometric data analysis for cortical nitrotyrosine expression (n = 10/group). *P < 0.05 vs. control. #P < 0.05 vs. db/db mice. C: representative photomicrograph of immunohistochemical stain for nitrotyrosine (arrows). a: Control. b: Control+nicotine. c: db/db. d: db/db+nicotine.

Fig. 6.

Effects of nicotine on Akt phosphorylation in diabetic mice. Diabetic mice had a significant increase in total Akt/PKB and p-Akt that was further increased by the administration of nicotine in the drinking water. The administration of nicotine to nondiabetic mice also induced a small increase in Akt/PKB and p-Akt/PKB. Representative results of Western blot analysis were obtained from 3 independent samples from each group. A: representative Western blot for Akt/PKB and p-Akt/PKB. B: densitometric data analysis for cortical Akt/PKB and p-Akt/PKB expression (n = 10/group). *P < 0.05 vs. control. #P < 0.05 vs. nicotine and db/db mice. **P < 0.05 vs. control, nicotine, and db/db.

Fig. 7.

Effects of nicotine on Akt/PKB phosphorylation in human mesangial cells. Nicotine increased Akt/PKB phosphorylation and had an additive effect with high glucose on Akt/PKB phosphorylation. A: representative Western blot for p-Akt/PKB and total Akt/PKB. B: densitometric data analysis for p-Akt/PKB (n = 4). *P < 0.05 vs. low glucose. **P < 0.05 vs. nicotine.