AJP - Heart Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 292: H76-H82, 2007. First published August 18, 2006; doi:10.1152/ajpheart.00693.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/H76    most recent
00693.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jaimes, E. A.
Right arrow Articles by Raij, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jaimes, E. A.
Right arrow Articles by Raij, L.

CALL FOR PAPERS
Cardiovascular-Renal Mechanisms in Health and Disease

Nicotine: the link between cigarette smoking and the progression of renal injury?

Edgar A. Jaimes,1,2,3 Run-Xia Tian,1 and Leopoldo Raij1,2,3

1Nephrology Section Veterans Affairs Medical Center, 2Renal Division, and the 3Vascular Biology Institute, Miller School of Medicine, University of Miami, Miami, Florida

Submitted 29 June 2006 ; accepted in final form 18 August 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cigarette smoke (CS) is the most important source of preventable morbidity and mortality in the United States. Recent clinical studies have suggested that, in addition to being a major cardiovascular risk factor, CS promotes the progression of kidney disease. The mechanisms by which CS promotes the progression of chronic kidney disease have not been elucidated. Here we demonstrate for the first time that human mesangial cells (MCs) are endowed with the nicotinic ACh receptors (nAChRs) {alpha}4, {alpha}5, {alpha}7, beta2, beta3, and beta4. Studies performed in other cell types have shown that these nAChRs are ionotropic receptors that function as agonist-regulated Ca2+ channels. Nicotine induced MC proliferation in a dose-dependent manner. At 10 –7 M, a concentration found in the plasma of active smokers, nicotine induced MC proliferation [control, 1,328 ± 50 vs. nicotine, 2,761 ± 90 counts/minute (cpm); P < 0.05] and increased the synthesis of fibronectin (50%), a critical matrix component involved in the progression of chronic kidney disease. We and others have shown that, in response to PKC activation, MC synthesize reactive oxygen species (ROS) via NADPH oxidase. In the current studies we demonstrate that PKC inhibition as well as diphenyleneiodonium and apocynin, two inhibitors of NADPH oxidase, prevented the effects of nicotine on MC proliferation and fibronectin production, hence establishing ROS as second messengers of the actions of nicotine. Furthermore, nicotine increased the production of ROS as assessed by 2',7'-dichlorofluorescein diacetate fluorescence [control, 184.4 ± 26 vs. nicotine, 281.5 ± 26 arbitrary fluorescence units (AFU); n = 5 experiments, P < 0.05]. These studies unveil previously unrecognized mechanisms that indict nicotine, a component of CS, as an agent that may accelerate and promote the progression of kidney disease.

glomerular mesangium; extracellular matrix; cell proliferation; reactive oxygen species

CIGARETTE SMOKING has been identified as the most important source of preventable morbidity and mortality in the United States (4, 27). In addition to being a risk factor for atherosclerosis and cancer, recent epidemiologic studies suggest that cigarette smoking increases the risk for progressive chronic kidney disease and accelerates the rate of progression of renal failure among patients with diabetes (31, 41, 45) and hypertension (3, 16, 28, 37). Clinical studies have indicated a correlation between smoking and the development of proteinuria in patients with polycystic kidney disease (5), as well a deterioration of renal function in patients with lupus nephritis (51), polycystic kidney disease, and glomerulonephritis (32, 46).

The mechanisms by which cigarette smoking accelerates the progression of chronic kidney disease have not been well studied. Studies from the laboratory of Jaimes and colleagues (17, 36) have demonstrated that stable compounds present in cigarette smoke produce endothelial dysfunction by increasing the vascular production of reactive oxygen species (ROS). Recent studies utilizing ambulatory blood pressure measurements have documented that cigarette smoking is associated with transitory increases in blood pressure (25, 29, 35) that have been attributed to direct stimulation of postganglionic sympathetic nerve endings (9) and related to nicotine since they are not observed with nicotine-free cigarettes (1). Nicotine administration leads to transitory elevations in blood pressure accompanied by a decrease in glomerular filtration rate and effective renal plasma flow in nonsmokers (12). In subjects with IgA nephropathy, a glomerular disease that characteristically affects the mesangium, nicotine administration is associated with increased urinary albumin excretion and reductions in glomerular filtration rate (38, 39).

Recent experimental studies (14) have demonstrated that nicotine is a potent stimulus for angiogenesis both in vivo and in vitro via activation of specific nicotinic ACh receptors (nAChRs). These nAChRs are ionotropic receptors that function as agonist-regulated Ca2+ channels and that are expressed by neuronal as well nonneuronal cells, including endothelial and vascular smooth muscle cells (14, 15). Whether nAChRs are expressed in glomerular cells is, however, not known. ROS are important mediators for the growth-related responses of a variety of cytokines and growth factors including ANG II (18), PDGF (47), and transforming growth factor-beta (TGF-beta) (22, 30). Nicotine has been shown to stimulate the production of ROS in lung and colon epithelial cells (10, 53). Whether ROS mediate the growth-promoting effects of nicotine in mesangial cells is, however, not known.1

The studies reported herein were designed to 1) determine whether mesangial cells possess nAChRs and 2) determine whether nicotine through activation of these receptors promotes mesangial cell proliferation and extracellular matrix production via ROS.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mesangial cell culture. Human mesangial cells were purchased from Cell Systems (Kirkland, WA), grown in CSC-Complete media (Cell Systems) supplemented with 10% fetal calf serum (Cell Systems). Cells were passed by trypsinization when confluent and used between the third and ninth passages.

Cell proliferation. [3H]thymidine incorporation was used as an index of cell proliferation as described (18). Briefly, cells were fasted for 72 h in maintenance media (Cell Systems) and stimulated for 24 h with nicotine (10–10 M to 10–7 M) or PDGF (10 ng/ml). Four hours before being harvested, cells were pulsed with [3H]thymidine (1 µCi/ml). At the end of this incubation period, cells were washed three times with PBS; protein was precipitated with 1 ml TCA 10% for 5 min and solubilized in 1 ml 0.5 N NaOH-0.1% SDS. Duplicate aliquots (0.5 ml) were removed and counted in a liquid scintillation counter and results expressed as counts per minute (cpm) per million cells.

Western blot analysis. Western blot analysis was performed as previously described (20, 21). Cells were fasted for 72 h in maintenance media (Cell Systems) and stimulated for 24 h with nicotine (10–7). Cell homogenates were washed once with PBS and resuspended in 300 µl homogenization buffer [containing (in mM) 50 Tris·HCl (pH 7.6), 100 NaCl, 2 EDTA, 2 EGTA, 1 DTT, and 1 PMSF and 1% Triton X-100] and incubated on ice for 30 min. Thereafter, lysates were centrifuged for 30 min at 10,000 g at 4°C. Supernatants were collected and the protein content was determined by Bio-Rad. Thirty micrograms of protein were separated by SDS-PAGE (6% acrylamide gel) and transferred to a nitrocellulose membrane. Blots were incubated overnight with one of the following antibodies: mouse anti-{alpha}2-nAChR MAbs (Sigma-Aldrich), anti-{alpha}3-nAChR MAbs (Santa Cruz Biotechnology), anti-{alpha}4-nAChR MAbs (Sigma-Aldrich), anti-{alpha}5-nAChR MAbs (Sigma-Aldrich), anti-{alpha}7-nAChR MAbs (Sigma-Aldrich), anti-beta2-nAChR MAbs (Sigma-Aldrich), anti-beta3-nAChR MAbs (Santa Cruz Biotechnology), anti-beta4-nAChR MAbs (Santa Cruz Biotechnology), polyclonal anti-fibronectin antibody (Sigma-Aldrich), and polyclonal anti-actin antibody (Santa Cruz Biotechnology). After being washed, the blots were incubated with goat anti-rabbit antibody (Santa Cruz Biotechnology) for 1 h, and the signal was detected by luminol chemiluminescence.

mRNA isolation and real-time quantitative RT-PCR. Fibronectin mRNA expression was determined by real-time PCR. Total RNA was isolated by utilizing the RNeasy Mini Kit (Qiagen, Valencia, CA). A 5-µg aliquot of total RNA was used for cDNA synthesis using the Superscript preamplification system (Life Technologies). Primers and probes for fibronectin were designed using Primer Express software 101 (ABI). As an active reference, endogenous 18S ribosomal RNA (r18S) was amplified by using specific primers and probes labeled with VIC (ABI). The comparative threshold cycle (CT) method was used for relative quantification and statistical analysis. A unit difference in cycle value represents a twofold change in mRNA abundance.

Flow cytometry measurement of ROS. ROS generation was measured by using flow cytometric analysis of 2',7'-dichlorofluorescein (DCF) diacetate (DCF-DA)-loaded cells as described by others (43). Briefly, human mesangial cells were grown to 80% confluence in complete media (Cell Systems), made quiescent for 48 h in maintenance media (Cell Systems), and stimulated with nicotine (10–7 M) with and without the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10–6 M). Cells were then incubated in the dark with DCF-DA (Molecular Probes) at a concentration of 7.5 µg/ml for 10 min at 37°C and scraped and resuspended in Hanks' solution. Fluorescence was measured by using a flow cytometer (FACSort, Becton-Dickinson). The mean DCF fluorescence intensity was measured using 480-nm excitation and 540-nm emission settings. Fluorescence was expressed in arbitrary fluorescence units (AFU).

Statistical analysis. Data are expressed as means ± SE. For statistical comparisons involving two groups, an unpaired Student's t-test was used, whereas for comparisons involving more than two groups, ANOVA (StatView, BrainPower, Calabasas, CA) was used. Significance was considered when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Human mesangial cells express nAChRs. We first determined whether human mesangial cells express nAChRs as assessed by Western blot analysis. As shown in Fig. 1, we were able to detect strong expression of several nAChR subunits in cultured human mesangial cells. We detected the presence of the nAChR subunits {alpha}4, {alpha}5, {alpha}7, beta2, beta3, and beta4 in these cells. We were, however, not able to detect significant expression of the {alpha}2 or {alpha}3 nAChR subunits. These findings demonstrate that under basal conditions, human mesangial cells express several subtypes of nAChRs.


Figure 1
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 1. Expression of nicotine ACh receptor (nAChR) subunits in human mesangial cells. Western blot analysis demonstrated the presence of the subunits {alpha}4, {alpha}5, {alpha}7, beta2, beta3, and beta4 under basal conditions in subconfluent human mesangial cells.

 
Nicotine increases mesangial cell proliferation via nAChR activation. To determine the effects of nicotine on mesangial cell proliferation, we exposed human mesangial cells to nicotine (10–10 to 10–7 M) or PDGF (10 ng/ml). Mesangial cell proliferation was assessed by [3H]thymidine incorporation. As shown in Fig. 2, stimulation with nicotine resulted in a dose-dependent increase in mesangial cell proliferation. Based on these results, we performed all subsequent experiments utilizing a nicotine concentration of 10–7 M, which is similar to the concentrations of nicotine found in the plasma of active smokers (50) and resulted in a proliferative response that was ~50% of that obtained with the potent mitogen PDGF: control, 932 ± 55; nicotine, 1,319 ± 82 (P < 0.05 vs. control); and PDGF, 1,836 ± 160 cpm (n = 3 experiments in duplicate; P < 0.05 vs. control, and P < 0.05 vs. nicotine).


Figure 2
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 2. Nicotine (10–7 to 10–10 M) induced mesangial cell proliferation in a dose-dependent manner. Results are expressed as means ± SE; n = 3 experiments in duplicate. *P < 0.05 vs. control. cpm, Counts/minute.

 
To determine the role of nAChR signaling on the pro-proliferative effects of nicotine, we treated human mesangial cells with nicotine (10–7 M) with and without the nonspecific nAChR blocker hexamethonium (10–4 M) (15) or the muscarinic receptor blocker atropine (10–7 M) (14, 15). As shown in Fig. 3, the nAChR blocker hexamethonium but not the muscarinic receptor blocker atropine prevented the effects of nicotine on mesangial cell proliferation, suggesting that these effects are mediated via nAChR activation. To determine the specific nAChRs involved, human mesangial cells were treated with nicotine (10–7 M) with and without the nAChR blockers {alpha}-bungarotoxin ({alpha}7-nAChR antagonist, 10–7 to 10–9 M) (15), {alpha}-lobeline ({alpha}4/beta2 nAChR antagonist, 10–6 to 10–8 M) (15), dihydro-beta-erythroidine ({alpha}3/beta2 and {alpha}4/beta2 nAChR antagonist, 10–6 to 10–8 M) (15). As shown in the Fig. 4, pretreatment with these nAChR antagonists prevented the effects of nicotine on mesangial cell proliferation. Stimulation of the {alpha}7-nAChR has been shown to mediate the proliferative effects of nicotine in vascular endothelial cells (15). Our findings suggest that signaling via the nicotinic receptors {alpha}7-nAChR, {alpha}4-nAChR, and beta2-nAChR is required for the pro-proliferative effects of nicotine on mesangial cells.


Figure 3
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 3. Nicotine-induced mesangial cell proliferation is prevented by the nonspecific nAChR blocker hexamethonium (10–4 M) but not by the muscarinic receptor blocker atropine (10–7 M). Results are expressed as means ± SE; n = 3 experiments in duplicate. *P < 0.05 vs. control.

 

Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 4. Nicotine-induced mesangial cell proliferation is prevented by the nicotine receptor blockers bungarotoxin (10–7 to 10–9 M), {alpha}-lobeline (10–6 to 10–8 M), and dihydro-beta-erythroidine (10–6 to 10–8 M). Results are expressed as means ± SE; n = 3 experiments in duplicate. *P < 0.05 vs. control; #P < 0.05 vs. nicotine.

 
Nicotine increases fibronectin production. To determine whether in addition to increasing mesangial cell proliferation nicotine increases extracellular matrix expression, we measured fibronectin protein expression in human glomerular mesangial cells exposed to nicotine (10–7 M). As shown in Fig. 5, nicotine induced a significant increase in fibronectin protein expression. To determine whether transcriptional mechanisms were involved, we measured fibronectin gene expression by real-time RT-PCR in human glomerular mesangial cells exposed to nicotine (10–7 M) with and without the nicotine receptor blocker hexamethonium (10–4 M). Nicotine induced a fivefold increase in fibronectin mRNA expression: control {Delta}CT, 11.37 ± 2.04 vs. nicotine {Delta}CT, 8.75 ± 1.25; n = 6 experiments, P < 0.05 that was prevented by the nicotine receptor blocker hexamethonium: {Delta}CT, 12.19 ± 2.23, n = 3 experiments, P = not significant (NS) vs. control.


Figure 5
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 5. Nicotine increases fibronectin production in human mesangial cells. The effects of nicotine fibronectin production by mesangial cells are prevented by the NADPH oxidase inhibitors diphenyleneiodonium (DPI; 10–5 M), apocynin (10–4 M), and catalase (800 U/ml). A: representative Western blot analysis for fibronectin and actin that was used to control for unequal loading: control (lane 1), nicotine (lane 2), nicotine + DPI (lane 3), nicotine + apocynin (lane 4), and nicotine + catalase (lane 5). B: densitometry data analysis performed after reprobing the blot with an {alpha}-actin antibody. Results are expressed as means ± SE; n = 3–6 experiments in duplicate. *P < 0.05 vs. control. OD, optical density.

 
ROS mediate nicotine-induced mesangial cell proliferation and fibronectin production. We and others have demonstrated that ROS play a major role as intracellular mediators of growth-related responses to growth factors and cytokines, such as ANG II, PDGF, and TGF-beta. To investigate whether NADPH oxidase-derived ROS participate in the growth-promoting effects of nicotine, human mesangial cells were exposed to DPI, a compound that by tightly binding to flavoproteins is an effective inhibitor of flavin-containing oxidases, such as NADPH oxidase (10–5 M) (18) or apocynin, a specific NADPH oxidase inhibitor (10–4 M) (17) before nicotine. Treatment with DPI or apocynin prevented the increases in mesangial cell proliferation in response to nicotine, demonstrating that NADPH oxidase-derived ROS are important intracellular signals responsible for nicotine-induced mesangial cell proliferation (Fig. 6). To assess the effects of these inhibitors under basal conditions, human mesangial cells were treated with DPI or apocynin for 24 h, and cell proliferation was assessed by [3H]thymidine incorporation. Treatment with DPI but not with apocynin reduced [3H]thymidine incorporation in unstimulated cells: control, 397 ± 136; DPI, 206 ± 108 (P < 0.05 vs. control); and apocynin 492 ± 172 cpm/million cells (n = 3 experiments). Pretreatment with the cell-permeable superoxide scavenger Tiron (5 mM) (21) prevented the effects of nicotine on mesangial cell proliferation, further suggesting a role for ROS on the growth-promoting effects of nicotine.


Figure 6
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 6. Nicotine-induced mesangial cell proliferation is prevented by the NADPH oxidases inhibitors DPI and apocynin as well as by catalase and Tiron. Results are expressed as means ± SE; n = 3 experiments in duplicate. *P < 0.05 vs. control; #P < 0.05 vs. control.

 
Superoxide anion produced as a result of increased NADPH oxidase activity rapidly dismutates to H2O2 spontaneously or via superoxide dismutase. As shown in Fig. 6, catalase (800 U/ml) (18), an enzyme that decomposes H2O2, inhibited nicotine-stimulated mesangial cell proliferation, suggesting that H2O2 is the main ROS responsible for nicotine-stimulated mesangial cell proliferation. Treatment of unstimulated cells with catalase did not result in significant reductions in mesangial cell proliferation: control, 397 ± 136 vs. catalase, 451 ± 27 cpm/million cells (n = 3 experiments, P = NS).

To determine the role of ROS on nicotine-induced ECM deposition, human mesangial cells were stimulated with nicotine (10–7 M) with and without the NADPH oxidase inhibitors DPI (10–5 M) or apocynin (10–4 M). Treatment with any of these two NADPH oxidase inhibitors prevented the effects of nicotine on fibronectin protein expression, suggesting that NADPH oxidase-derived ROS mediate the effects of nicotine on fibronectin production (Fig. 5). Similarly, treatment with catalase prevented the effects of nicotine on fibronectin production, suggesting that H2O2 is a critical mediator of the effects of nicotine on fibronectin production (Fig. 5).

To determine the effects of nicotine on ROS production in human mesangial cells, we used DCF-DA fluorescence as a marker of ROS generation. Preliminary experiments suggested that nicotine increased ROS generation in a time-dependent manner that peaked at 1 h and returned to baseline after 4 h (not shown). Based on these pilot studies, subsequent experiments were performed after 1-h incubation. Nicotine stimulation (10–7 M) resulted in a significant increase in ROS generation as assessed by DCF fluorescence: control, 184.4 ± 26 vs. nicotine, 281.5 ± 26 AFU (n = 5 experiments, P < 0.05) (Fig. 7). To determine whether nicotine increased ROS generation via NADPH oxidase, human mesangial cells were incubated with nicotine with and without the NADPH oxidase inhibitor DPI (10–5). NADPH oxidase inhibition with DPI resulted in complete inhibition of nicotine-stimulated ROS generation: nicotine, 328.9 ± 8 vs. nicotine + DPI, 142.9 ± 20 AFU (n = 3 experiments, P < 0.05).


Figure 7
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 7. Nicotine increases reactive oxygen species production as assessed by flow cytometric analysis. Human mesangial cells were stimulated with nicotine (10–7 M), stained with 2',7'-dichlorofluorescein diacetate and analyzed by flow cytometry. Reactive oxygen species are expressed as a histogram of fluorescence. Dotted line histogram represents unstimulated mesangial cells; solid line histogram represents nicotine-stimulated cells.

 
PKC activation has been shown to be involved in cell growth responses in response to a variety of stimuli, at least in part by participating in the activation of NADPH oxidase (18). To determine the role of PKC activation on the effects of nicotine in mesangial cells, we stimulated human mesangial cells with nicotine (10–7 M) with and without the PKC inhibitor calphostin C (10–6 M) (18). PKC inhibition with calphostin C prevented the effects of nicotine on mesangial cell proliferation (Fig. 8) and fibronectin production (Fig. 9), suggesting that PKC activation is required for these effects. Similar findings were obtained utilizing H-7 (10–6 M) (18), a PKC inhibitor chemically distinct from calphostin C (Fig. 8).


Figure 8
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 8. Nicotine-induced mesangial cell proliferation is inhibited by the PKC inhibitor calphostin C (Cal C) and H-7, the ERK1/2 inhibitor PD-98059 but not the p38 MAPK inhibitor SB-202190. Results are expressed as means ± SE; n = 3 experiments in duplicate. *P < 0.05 vs. control.

 

Figure 9
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 9. Nicotine-induced fibronectin production is inhibited by the PKC inhibitor calphostin C and the MAPK inhibitor PD-98059. A: representative Western blot analysis for fibronectin and actin that was used to control for unequal loading: control (lane 1), nicotine (lane 2), nicotine + calphostin C (lane 3), and nicotine + PD-98050 (lane 4). B: densitometry data analysis performed after reprobing the blot with an {alpha}-actin antibody (n = 3–6 experiments, *P < 0.05 vs. control).

 
MAPKs activation has been shown to participate in growth-related responses to growth factors and cytokines including ANG II (18), TGF-beta (13), and PDGF (6). To determine the role of MAPK-1 on the growth-promoting effects of nicotine, we stimulated mesangial cells with nicotine (10–7 M) with and without the ERK1/2 inhibitor PD-98059 (10–6 M) or the p38 MAPK inhibitor SB-202190 (10–6 M) (48). ERK1/2, but not p38 MAPK, inhibition prevented the effects of nicotine on mesangial cell proliferation (Fig. 8), suggesting a critical role for ERK1/2 on the growth-promoting effects of nicotine in mesangial cells. Furthermore, ERK1/2 inhibition also prevented the effects of nicotine on fibronectin production in mesangial cells (Fig. 9).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In these studies we have identified, to the best of our knowledge for the first time, the presence of functionally active nAChRs in human mesangial cells and demonstrated that nicotine, at concentrations similar to those found in the plasma of smokers (50), promotes mesangial cell proliferation and upregulates critical molecules involved in extracellular matrix production.

Recent epidemiologic studies demonstrated that cigarette smoking increases the risk for progressive chronic kidney disease and has been shown to accelerate the rate of progression of renal failure among patients with diabetes (31, 41, 45) and hypertension (3, 16, 28, 37). The role of smoking in primary renal diseases is less known, but clinical studies have indicated a correlation with the development of proteinuria in patients with polycystic kidney disease (5) and deterioration of renal function in patients with various nephritides, including lupus nephritis (51) and IgA nephropathy, a glomerular disease that predominantly affects the glomerular mesangium (32, 46).

The mechanisms by which cigarette smoking is a risk factor for progressive chronic kidney disease have not been established. Clinical and experimental evidence suggests that smoking has significant systemic and renal hemodynamic effects. Studies utilizing ambulatory blood pressure measurements have shown that cigarette smoking is associated with significant, albeit transitory, increases in blood pressure in subjects with normotension (29), a prior history of hypertension (25), diabetes (35), and primary glomerular disease (33, 38). Recent population-based, cross-sectional studies have demonstrated that the risk in developing hypertension is higher among smokers, especially for men >60 yr old (11). The transitory increases in blood pressure observed in smokers have been attributed, at least in part, to direct stimulation of postganglionic sympathetic nerve endings, leading to an increase of plasma concentrations of norepinephrine and epinephrine (9). It has been postulated that these effects are related to nicotine since they are not observed with nicotine-free cigarettes (1). Moreover, in subjects with IgA nephropathy, nicotine administration is associated with increased urinary albumin excretion and reductions in glomerular filtration rate compared with healthy volunteers (38, 39).

Nicotine is a major component of tobacco and responsible in large part for the psychoactive and addictive effects of cigarette smoking via its action in the central nervous system (34). In addition, recent studies have demonstrated that nicotine also has significant biological effects outside of the central nervous system. In the vasculature, nicotine has been shown to stimulate angiogenesis and promote atherosclerosis (14). The effects of nicotine in the vasculature appear to be mediated by nonneuronal nAChRs. These nAChRs are ionotropic receptors that function as agonist-regulated Ca2+ channels and that are expressed by neuronal as well nonneuronal cells, including endothelial and vascular smooth muscle cells (8, 15, 24, 26, 52). We have now identified the presence of functionally active nAChRs in human mesangial cells, suggesting that nicotine may have important biological actions in the glomerulus. The nAChRs have a pentameric structure consisting of five nAChR subunits organized around a central channel and can be either homo- or hetero-oligomeric (7). The expression of the {alpha}7 subunit results in the formation of nAChRs that can be blocked by nanomolar concentrations of {alpha}-bungarotoxin because of the high affinity of the {alpha}7 subunit for this compound (23). Our results suggest that human mesangial cells express hetero-oligomeric nAChRs containing the {alpha}7 subunit, given the inhibitory effects with nanomolar concentrations of {alpha}-bungarotoxin, and also containing the subunits {alpha}4 and beta2, given the inhibitory effects of {alpha}-lobeline and dihydro-beta-erythroidine.

In addition to inducing vascular smooth muscle cell proliferation, nicotine has also been shown to increase extracellular matrix deposition by several cell types. Nicotine increases fibronectin production in lung fibroblasts in vivo and in vitro (40), and in dermal fibroblasts, nicotine, acting via the {alpha}3 nAChR, significantly increases the expression of type 1 collagen, elastin, and matrix metalloproteinase-1 (2). Here we demonstrate that nicotine is a powerful stimulus for mesangial cell proliferation and fibronectin production, therefore providing a potential mechanism for the deleterious effects of cigarette smoking on the progression of chronic kidney disease, particularly since our experiments were performed in human mesangial cells and utilized concentrations of nicotine similar to those found in active smokers (50).

We have previously demonstrated that ROS mediate mesangial cell proliferation and hypertrophy in response to ANG II (18). In the current studies, apocynin and DPI, two inhibitors of NADPH oxidase, prevented the effects of nicotine on mesangial cell proliferation and fibronectin production, suggesting that NADPH oxidase-derived ROS mediate the growth-related effects of nicotine in human mesangial cells. Moreover, our flow cytometry assays demonstrate that nicotine increases the production of ROS via NADPH oxidase. In recent studies (17), we have also demonstrated that stable compounds, such as acrolein, present in cigarette smoke increase the endothelial production of ROS via NADPH oxidase activation.

We and others have shown that PKC activation is involved in NADPH oxidase activation in several cell types, including mesangial cells and vascular smooth muscle cells (18, 44). In our current studies, PKC inhibition inhibited mesangial cell proliferation and fibronectin production in response to nicotine, suggesting that PKC activation is required for the growth-promoting effects of nicotine, probably by participating in the activation of NADPH oxidase.

ROS, including O2–, have been shown to promote activation of MAPKs (42, 49), a process that is associated with growth-related responses (6, 18). ERK1 and ERK2, the best studied MAPKs, are involved in cellular growth and differentiation in response to a variety of stimuli, including ANG II (18), PDGF (6), and TGF-beta (13). In our studies, specific ERK1/2 inhibition with PD-98059 prevented mesangial cell proliferation and fibronectin production in response to nicotine, suggesting that ERK1 and ERK2 activation is required for the growth-promoting effects of nicotine. In contrast, p38 MAPK inhibition did not significantly inhibit the growth-promoting effects of nicotine, suggesting that p38 MAPK does not play a significant role on these effects.

In summary, our past (17) and, particularly, present studies unveil novel mechanisms by which cigarette smoke may accelerate and promote the progression of chronic kidney disease.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by a Merit Review Award from the Veterans Affairs Administration, a research grant from the Flight Attendants Research Institute, and a National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-069372.


   ACKNOWLEDGMENTS
 
Results from these studies were presented in part at the Ninth Cardiovascular-Kidney Interactions in Health and Disease Meeting, held at Amelia Island Plantation, Florida, May 26–29, 2006. We thank Kenya Essix and Judy Hunter for secretarial support and Jessica Nigro for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. A. Jaimes, VA Medical Center, 1201 NW 16th St., Renal Section, Rm. A-1009, Miami FL, 33125 (e-mail: ejaimes{at}med.miami.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This paper was presented at the 9th Cardiovascular-Kidney Interactions in Health and Disease Meeting at Amelia Island Plantation, Florida, on May 26–29, 2006. Back


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aronow WS, Dendinger J, and Rokaw SN. Heart rate and carbon monoxide level after smoking high, low-, and non-nicotine cigarettes. A study in male patients with angina pectoris. Ann Intern Med 74: 697–702, 1971.[Abstract/Free Full Text]
  2. Arredondo J, Hall LL, Ndoye A, Nguyen VT, Chernyavsky AI, Bercovich D, Orr-Urtreger A, Beaudet AL, and Grando SA. Central role of fibroblast alpha3 nicotinic acetylcholine receptor in mediating cutaneous effects of nicotine. Lab Invest 83: 207–225, 2003.[Web of Science][Medline]
  3. Bleyer AJ, Shemanski LR, Burke GL, Hansen KJ, and Appel RG. Tobacco, hypertension, and vascular disease: risk factors for renal functional decline in an older population. Kidney Int 57: 2072–2079, 2000.[CrossRef][Web of Science][Medline]
  4. Boyle P. Cancer, cigarette smoking and premature death in Europe: a review including the Recommendations of European Cancer Experts Consensus Meeting, Helsinki, October 1996. Lung Cancer 17: 1–60, 1997.[CrossRef][Web of Science][Medline]
  5. Chapman AB, Johnson AM, Gabow PA, and Schrier RW. Overt proteinuria and microalbuminuria in autosomal dominant polycystic kidney disease. J Am Soc Nephrol 5: 1349–1354, 1994.[Abstract]
  6. Choudhury GG, Karamitsos C, Hernandez J, Gentilini A, Bardgette J, and Abboud HE. PI-3-kinase and MAPK regulate mesangial cell proliferation and migration in response to PDGF. Am J Physiol Renal Physiol 273: F931–F938, 1997.[Abstract/Free Full Text]
  7. Gotti C, and Clementi F. Neuronal nicotinic receptors: from structure to pathology. Prog Neurobiol 74: 363–396, 2004.[CrossRef][Web of Science][Medline]
  8. Grando SA, Horton RM, Pereira EF, Diethelm-Okita BM, George PM, Albuquerque EX, and Conti-Fine BM. A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes. J Invest Dermatol 105: 774–781, 1995.[CrossRef][Web of Science][Medline]
  9. Grassi G, Seravalle G, Calhoun DA, Bolla GB, Giannattasio C, Marabini M, Del Bo A, and Mancia G. Mechanisms responsible for sympathetic activation by cigarette smoking in humans. Circulation 90: 248–253, 1994.
  10. Guo J, Chu M, Abbeyquaye T, and Chen CY. Persistent nicotine treatment potentiates amplification of the dihydrofolate reductase gene in rat lung epithelial cells as a consequence of Ras activation. J Biol Chem 280: 30422–30431, 2005.[Abstract/Free Full Text]
  11. Halimi JM, Giraudeau B, Vol S, Caces E, Nivet H, and Tichet J. The risk of hypertension in men: direct and indirect effects of chronic smoking. J Hypertens 20: 187–193, 2002.[CrossRef][Web of Science][Medline]
  12. Halimi JM, Philippon C, and Mimran A. Contrasting renal effects of nicotine in smokers and non-smokers. Nephrol Dial Transplant 13: 940–944, 1998.[Abstract/Free Full Text]
  13. Hartsough MT, and Mulder KM. Transforming growth factor beta activation of p44mapk in proliferating cultures of epithelial cells. J Biol Chem 270: 7117–7124, 1995.[Abstract/Free Full Text]
  14. Heeschen C, Jang JJ, Weis M, Pathak A, Kaji S, Hu RS, Tsao PS, Johnson FL, and Cooke JP. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nat Med 7: 833–839, 2001.[CrossRef][Web of Science][Medline]
  15. Heeschen C, Weis M, Aicher A, Dimmeler S, and Cooke JP. A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholine receptors. J Clin Invest 110: 527–536, 2002.[CrossRef][Web of Science][Medline]
  16. Horner D, Fliser D, Klimm HP, and Ritz E. Albuminuria in normotensive and hypertensive individuals attending offices of general practitioners. J Hypertens 14: 655–660, 1996.[CrossRef][Web of Science][Medline]
  17. Jaimes EA, DeMaster EG, Tian RX, and Raij L. Stable compounds of cigarette smoke induce endothelial superoxide anion production via NADPH oxidase activation. Arterioscler Thromb Vasc Biol 24: 1031–1036, 2004.[Abstract/Free Full Text]
  18. Jaimes EA, Galceran JM, and Raij L. Angiotensin II induces superoxide anion production by mesangial cells. Kidney Int 54: 775–784, 1998.[CrossRef][Web of Science][Medline]
  19. Jaimes EA, Galceran JM, and Raij L. Angiotensin II induces superoxide anion production by mesangial cells. Kidney Int 54: 775–784, 1998.[CrossRef][Web of Science][Medline]
  20. Jaimes EA, Nath KA, and Raij L. Hydrogen peroxide downregulates IL-1-driven mesangial iNOS activity: implications for glomerulonephritis. Am J Physiol Renal Physiol 272: F721–F728, 1997.[Abstract/Free Full Text]
  21. Jaimes EA, Tian RX, Pearse D, and Raij L. Up-regulation of glomerular COX-2 by angiotensin II: role of reactive oxygen species. Kidney Int 68: 2143–2153, 2005.[CrossRef][Web of Science][Medline]
  22. Jiang Z, Seo JY, Ha H, Lee EA, Kim YS, Han DC, Uh ST, Park CS, and Lee HB. Reactive oxygen species mediate TGF-beta1-induced plasminogen activator inhibitor-1 upregulation in mesangial cells. Biochem Biophys Res Commun 309: 961–966, 2003.[CrossRef][Web of Science][Medline]
  23. Lukas RJ, and Bencherif M. Heterogeneity and regulation of nicotinic acetylcholine receptors. Int Rev Neurobiol 34: 25–131, 1992.[Web of Science][Medline]
  24. Macklin KD, Maus AD, Pereira EF, Albuquerque EX, and Conti-Fine BM. Human vascular endothelial cells express functional nicotinic acetylcholine receptors. J Pharmacol Exp Ther 287: 435–439, 1998.[Abstract/Free Full Text]
  25. Mann SJ, James GD, Wang RS, and Pickering TG. Elevation of ambulatory systolic blood pressure in hypertensive smokers. A case-control study. JAMA 265: 2226–2228, 1991.[Abstract/Free Full Text]
  26. Maus AD, Pereira EF, Karachunski PI, Horton RM, Navaneetham D, Macklin K, Cortes WS, Albuquerque EX, and Conti-Fine BM. Human and rodent bronchial epithelial cells express functional nicotinic acetylcholine receptors. Mol Pharmacol 54: 779–788, 1998.[Abstract/Free Full Text]
  27. McBride PE. The health consequences of smoking. Cardiovascular diseases. Med Clin North Am 76: 333–353, 1992.[Web of Science][Medline]
  28. Mimran A, Ribstein J, DuCailar G, and Halimi JM. Albuminuria in normals and essential hypertension. J Diabetes Complications 8: 150–156, 1994.[CrossRef][Web of Science][Medline]
  29. Minami J, Ishimitsu T, and Matsuoka H. Effects of smoking cessation on blood pressure and heart rate variability in habitual smokers. Hypertension 33: 586–590, 1999.[Abstract/Free Full Text]
  30. Ohba M, Shibanuma M, Kuroki T, and Nose K. Production of hydrogen peroxide by transforming growth factor-beta 1. J Cell Biol 126: 1079–1088, 1994.[Abstract/Free Full Text]
  31. Orth SR. Smoking—a renal risk factor. Nephron 86: 12–26, 2000.[CrossRef][Web of Science][Medline]
  32. Orth SR, Stockmann A, Conradt C, Ritz E, Ferro M, Kreusser W, Piccoli G, Rambausek M, Roccatello D, Schafer K, Sieberth HG, Wanner C, Watschinger B, and Zucchelli P. Smoking as a risk factor for end-stage renal failure in men with primary renal disease. Kidney Int 54: 926–931, 1998.[CrossRef][Web of Science][Medline]
  33. Orth SR, Viedt C, and Ritz E. Adverse effects of smoking in the renal patient. Tohoku J Exp Med 194: 1–15, 2001.[CrossRef][Web of Science][Medline]
  34. Pontieri FE, Tanda G, Orzi F, and Di Chiara G. Effects of nicotine on the nucleus accumbens and similarity to those of addictive drugs. Nature 382: 255–257, 1996.[CrossRef][Medline]
  35. Poulsen PL, Ebbehoj E, Hansen KW, and Mogensen CE. Effects of smoking on 24-h ambulatory blood pressure and autonomic function in normoalbuminuric insulin-dependent diabetes mellitus patients. Am J Hypertens 11: 1093–1099, 1998.[CrossRef][Web of Science][Medline]
  36. Raij L, DeMaster EG, and Jaimes EA. Cigarette smoke-induced endothelium dysfunction: role of superoxide anion. J Hypertens 19: 891–897, 2001.[CrossRef][Web of Science][Medline]
  37. Regalado M, Yang S, and Wesson DE. Cigarette smoking is associated with augmented progression of renal insufficiency in severe essential hypertension. Am J Kidney Dis 35: 687–694, 2000.[Web of Science][Medline]
  38. Ritz E, Benck U, Franek E, Keller C, Seyfarth M, and Clorius J. Effects of smoking on renal hemodynamics in healthy volunteers and in patients with glomerular disease. J Am Soc Nephrol 9: 1798–1804, 1998.[Abstract]
  39. Ritz E, Benck U, and Orth SR. Acute effects of cigarette smoking on renal hemodynamics. Contrib Nephrol 130: 31–38, 2000.[Web of Science][Medline]
  40. Roman J, Ritzenthaler JD, Gil-Acosta A, Rivera HN, and Roser-Page S. Nicotine and fibronectin expression in lung fibroblasts: implications for tobacco-related lung tissue remodeling. FASEB J 18: 1436–1438, 2004.[Abstract/Free Full Text]
  41. Rossing P, Hougaard P, and Parving HH. Risk factors for development of incipient and overt diabetic nephropathy in type 1 diabetic patients: a 10-year prospective observational study. Diabetes Care 25: 859–864, 2002.[Abstract/Free Full Text]
  42. Sano M, Fukuda K, Sato T, Kawaguchi H, Suematsu M, Matsuda S, Koyasu S, Matsui H, Yamauchi-Takihara K, Harada M, Saito Y, and Ogawa S. ERK and p38 MAPK, but not NF-kappaB, are critically involved in reactive oxygen species-mediated induction of IL-6 by angiotensin II in cardiac fibroblasts. Circ Res 89: 661–669, 2001.[Abstract/Free Full Text]
  43. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, and Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res 91: 406–413, 2002.[Abstract/Free Full Text]
  44. Sharma P, Evans AT, Parker PJ, and Evans FJ. NADPH-oxidase activation by protein kinase C-isotypes. Biochem Biophys Res Commun 177: 1033–1040, 1991.[CrossRef][Web of Science][Medline]
  45. Stegmayr BG. A study of patients with diabetes mellitus (type 1) and end-stage renal failure: tobacco usage may increase risk of nephropathy and death. J Intern Med 228: 121–124, 1990.[Web of Science][Medline]
  46. Stengel B, Couchoud C, Cenee S, and Hemon D. Age, blood pressure and smoking effects on chronic renal failure in primary glomerular nephropathies. Kidney Int 57: 2519–2526, 2000.[Web of Science][Medline]
  47. Sundaresan M, Yu ZX, Ferrans VJ, Irani K, and Finkel T. Requirement for generation of H2O2 for platelet-derived growth signal transduction. Science 270: 296–299, 1995.[Abstract/Free Full Text]
  48. Tharaux PL, Chatziantoniou C, Fakhouri F, and Dussaule JC. Angiotensin II activates collagen I gene through a mechanism involving the MAP/ER kinase pathway. Hypertension 36: 330–336, 2000.[Abstract/Free Full Text]
  49. Tian W, Zhang Z, and Cohen DM. MAPK signaling and the kidney. Am J Physiol Renal Physiol 279: F593–F604, 2000.[Abstract/Free Full Text]
  50. Urakawa N, Nagata T, Kudo K, Kimura K, and Imamura T. Simultaneous determination of nicotine and cotinine in various human tissues using capillary gas chromatography/mass spectrometry. Int J Legal Med 106: 232–236, 1994.[CrossRef][Web of Science][Medline]
  51. Ward MM, and Studenski S. Clinical prognostic factors in lupus nephritis. The importance of hypertension and smoking. Arch Intern Med 152: 2082–2088, 1992.[Abstract/Free Full Text]
  52. Wessler I, Kirkpatrick CJ, and Racke K. The cholinergic 'pitfall': acetylcholine, a universal cell molecule in biological systems, including humans. Clin Exp Pharmacol Physiol 26: 198–205, 1999.[CrossRef][Web of Science][Medline]
  53. Ye YN, Liu ES, Shin VY, Wu WK, and Cho CH. The modulating role of nuclear factor-kappaB in the action of alpha7-nicotinic acetylcholine receptor and cross-talk between 5-lipoxygenase and cyclooxygenase-2 in colon cancer growth induced by 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone. J Pharmacol Exp Ther 311: 123–130, 2004.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CJASNHome page
S. R. Orth and S. I. Hallan
Smoking: A Risk Factor for Progression of Chronic Kidney Disease and for Cardiovascular Morbidity and Mortality in Renal Patients Absence of Evidence or Evidence of Absence?
Clin. J. Am. Soc. Nephrol., January 1, 2008; 3(1): 226 - 236.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/H76    most recent
00693.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jaimes, E. A.
Right arrow Articles by Raij, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jaimes, E. A.
Right arrow Articles by Raij, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.