Nicotine exposure and the progression of chronic kidney disease: role of the α7-nicotinic acetylcholine receptor

Abstract

Clinical studies have established the role of cigarette smoking as a risk factor in the progression of chronic kidney disease (CKD). We have shown that nicotine promotes mesangial cell proliferation and hypertrophy via nonneuronal nicotinic acetylcholine receptors (nAChRs). The α7-nAChR is one of the most important subunits of the nAChRs. These studies were designed to test the hypothesis that nicotine worsens renal injury in rats with 5/6 nephrectomy (5/6Nx) and that the α7-nAChR subunit is required for these effects. We studied five different groups: Sham, 5/6Nx, 5/6Nx + nicotine (Nic; 100 μg/ml dry wt), 5/6Nx + Nic + α7-nAChR blocker methyllicaconitine (MLA; 3 mg·kg(-1)·day(-1) sq), and Sham + Nic. Blood pressure was measured by the tail-cuff method, and urine was collected for proteinuria. After 12 wk, the rats were euthanized and kidneys were collected. We observed expression of the α7-nAChR in the proximal and distal tubules. The administration of nicotine induced a small increase in blood pressure and resulted in cotinine levels similar to those found in the plasma of smokers. In 5/6Nx rats, the administration of nicotine significantly increased urinary protein excretion (onefold), worsened the glomerular injury score and increased fibronectin (∼ 50%), NADPH oxidase 4 (NOX4; ∼100%), and transforming growth factor-β expression (∼200%). The administration of nicotine to sham rats increased total proteinuria but not albuminuria, suggesting direct effects on tubular protein reabsorption. These effects were prevented by MLA, demonstrating a critical role for the α7-nAChR as a mediator of the effects of nicotine in the progression of CKD.

Fig. 1.

Cortical expression of the α7-nicotinic acetylcholine receptors (nAChRs). A: representative Western blot for the α7-nAChR and β-actin, which was used to control for loading. B: densitometry data analysis for cortical α7-nAChR expression (n = 6–7 per group; *P < 0.05 vs. Sham). 5/6Nx, 5/6 nephrectomy; Nic, nicotine; MLA, methyllicaconitine; OD, optical density.

Fig. 2.

Cortical localization of the α7-nAChR. A: control slide incubated with primary antibody but without secondary antibody (×20). B: representative photomicrograph demonstrating expression of α7-nAChR in the proximal tubules as identified by Lotus tetragonolobus lectin (×20). C: representative photomicrograph of a slide showing expression of α7-nAChR in the distal tubules as identified by peanut agglutinin (×20). D: representative photomicrograph demonstrating lack of expression of the α7-nAChR in the intrarenal vasculature (×20).

Fig. 3.

Urinary protein excretion. Urinary protein excretion at baseline and during the 12 wk following surgery. Urine was collected biweekly, and protein excretion was adjusted against urinary creatinine excretion (*P < 0.05 vs. all other groups; #P < 0.05 vs. 5/6Nx; †P < 0.05 vs. 5/6Nx + Nic).

Fig. 4.

Glomerular injury score. Glomerular injury score was assessed in all groups and the results presented as percent and severity of damaged glomeruli on a 0+ to 4+ scale where 0 is no damage [*P < 0.05 vs. 5/6Nx, 5/6Nx + Nic, and 5/6Nx + Nic + MLA; #P < 0.05 vs. 5/6Nx; †P < 0.05 vs. 5/6Nx + Nic].

Fig. 5.

Cortical fibronectin expression. A, B, and C: representative Western blot for fibronectin and β-actin, which was used to control for loading. D: densitometry data analysis for cortical fibronectin expression (n = 6–7 per group; *P < 0.05 vs. 5/6 Nx and 5/6Nx + Nic; #P < 0.05 vs. 5/6Nx; †P < 0.05 vs. 5/6Nx + Nic).

Fig. 6.

Fibronectin expression by immunofluorescence. A: control negative slide incubated with primary antibody but without secondary antibody (×20). B: representative photomicrograph of a sham-operated rat showing low interstitial expression of fibronectin (×20). C: representative photomicrograph from a 5/6Nx rat showing increased interstitial expression of fibronectin (×20). D: representative photomicrograph from a 5/6Nx + Nic rat showing increased interstitial and glomerular expression of fibronectin (×20). E: representative photomicrograph from a 5/6Nx + Nic + MLA rat showing reduced expression of fibronectin compared with 5/6Nx + Nic (D; ×20). F: representative photomicrograph from a Sham + Nic rat showing no change in the expression of fibronectin (×20).

Fig. 7.

Transforming growth factor (TGF)-β expression in kidney lysates. Administration of nicotine to 5/6Nx rats resulted in a twofold increase in TGF-β as assessed by ELISA in kidney cortex lysates. MLA prevented the increase in TGF-β suggesting that these effects are mediated by the α7-nAChR (*P < 0.05 vs. all other groups).

Fig. 8.

Nitrotyrosine expression. A: representative western blot for nitrotyrosine and β-actin, which was used to control for loading. B: densitometry data analysis for cortical nitrotyrosine expression (n = 6–7 per group; *P < 0.05 vs. 5/6Nx + Nic and 5/6Nx + Nic + MLA; #P < 0.05 vs. 5/6Nx).

Fig. 9.

NADPH oxidase 4 (NOX4) expression. A: representative Western blot for NOX4 and β-actin, which was used to control for loading. B: densitometry data analysis for cortical NOX4 expression (n = 6–7 per group; *P < 0.05 vs. Sham, 5/6Nx, and 5/6Nx + Nic; †P < 0.05 vs. 5/6Nx + Nic).

Fig. 10.

Urinary isoprostanes. Administration of nicotine to 5/6Nx rats resulted in a twofold increase in TGF-β as assessed by ELISA in kidney cortex lysates. MLA prevented the increase in TGF-β, suggesting that these effects are mediated by the α7-nAChR (*P < 0.05 vs. all other groups).