E-cigarette Smoke Exposure Elevates Renal MR Expression and Induces BP Elevation

Abstract

Background:

E-cigarette use increases the risk of blood pressure (BP) elevation in users, though the underlying mechanisms remain unclear. The mineralocorticoid receptor (MR) is known to play an important role in the regulation of renal electrolyte balance and BP, and is protected by 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). However, little is known about the effects of renal MR on e-cigarette-induced BP elevation.

Methods:

C57BL/6J male mice were exposed to aerosol PBS, e-cigarettes without nicotine, and e-cigarettes with 2.4% nicotine, with concurrent exposure to either a vehicle or the MR antagonist eplerenone.

Results:

Inhalation of e-cigarettes with nicotine markedly induced renal MR abundance and increased mean arterial BP in response to elevated plasma nicotine levels in C57BL/6J mice. Induction of MR by e-cigarettes was correlated with the reduction of 11β-HSD2 and activation of glycogen synthase kinase-3β phosphorylation (pSer⁹GSK3β) within the kidneys. In contrast, e-cigarettes increased the urinary ratio of corticosterone to 11-dehydrocorticosterone, reduced urinary sodium content, and elevated renal ENaC expression. However, inhaling e-cigarettes without nicotine did not affect these metabolic parameters. Treatment with eplerenone normalized BP, reversed urinary metabolic profiles, and reduced ENaC by inhibiting renal pSer⁹GSK3β in nicotine e-cigarette-exposed mice. In mouse renal CCD M1 cells, aerosol nicotine from e-cigarettes increased MR while decreasing 11β-HSD2, and these effects are likely via activation of pSer⁹GSK3β through a nicotinic receptor-mediated mechanism.

Conclusions:

Our findings highlight the potential renal health damage from e-cigarettes and suggest that aerosol nicotine-mediated induction of MR action in the kidneys may contribute to electronic smoking-induced BP elevation.

Graphical Abstract

Introduction

Tobacco smoking remains the leading cause of global health problems, contributing to over 7.7 million premature deaths annually worldwide.¹ In efforts to promote smoking cessation, e-cigarettes have emerged as substitutes, leading to a sharp rise in usage among both smokers and non-smokers.^2,3 The growing popularity of e-cigarettes has raised concerns about their potential health risks due to the lack of evidence and information regarding their long-term effects and safety.⁴ Clinical studies have shown that the use of e-cigarette containing nicotine can acutely elevate mean arterial pressure in smokers as well as in young non-smokers.^5–7 Cross-sectional population studies provide evidence that current users of e-cigarettes are associated with increased odds of hypertension.^8,9 Animal model studies report that acute exposure to e-cigarette vapor alters cardiovascular function and raises blood pressure (BP).¹⁰ Similarly, chronic exposure to e-cigarette vapors causes a progressive and steady rise in systolic BP in rodent models.^11–13 These studies imply that e-cigarette users may be more susceptible to the risk of hypertension, although the causal relationship remains unclear.

The harmful effects of e-cigarette smoking on BP homeostasis in nonsmokers have been reported to be primarily due to inhalation of nicotine aerosols,^14,15 similar to those observed in rodent models after e-cigarette exposure. This is highly relevant since almost all e-cigarette products sold in the market contain nicotine, suggesting that inhaling nicotine likely play in important role in linking e-cigarette use to the risk of hypertension. Nicotine exposure promotes the progress of hypertension caused by smoking and can also induce hypercortisolemia, suggesting that elevated glucocorticoid (GC) tone contributes to smoking-induced hypertension. Endogenous GC-induced hypertension is recognized as being mediated by enhancing sodium reabsorption and is associated with abnormal activation of renal mineralocorticoid receptors (MR).¹⁶ The MR is a ligand-activated transcriptional factor with similar binding affinities for both aldosterone and cortisol and its principal function in the kidney is to regulate electrolyte balance and BP through sodium transport.^17–21 Inappropriate activation of MR by GCs in the kidneys can induce sodium retention and hypertension,^22,23 underlining the importance of MR action in the pathogenesis of hypertension.

The in vivo selectivity of the MR for its cognate ligand aldosterone is protected from GCs by 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which converts active cortisol to inactive cortisone, even though circulating GC levels are 100 to 1000 times higher than those of aldosterone.²⁴ 11β-HSD2 is known to colocalize with MR in the distal nephron of the kidneys, as well as in the brains and vascular wall, the sites of mineralocorticoid action.²⁵ Lack of 11β-HSD2 can cause sodium retention and hypertension as a result of GC-induced activation of renal MR, which can be blunted by MR antagonists.^26,27 This enzyme thus determines the pre-receptor regulation of MR signaling for BP control. The response of MR to its ligand steroids is modified by glycogen synthase kinase-3β (GSK3β), a crucial mediator for ligand-dependent gene transcription, and is activated by GCs and aldosterone.^28,29 Increased serine/threonine phosphorylation levels of GSK3β are positively correlated with aldosterone-induced activation of MR gene expression in renal cells.³⁰ GSK3β has also emerged as a negative regulator of C/EBPβ and its phosphorylation site has been identified in the putative consensus sequence region of C/EBPβ.^31,32 On the other hand, the levels of pSer⁹GSK3β are reported to be increased,³³ whereas the transcriptional levels of C/EBPβ are decreased in the bronchial epithelial cells of smokers.³⁴ However, whether inhaling nicotine from e-cigarette vapors might modulate renal MR action and GSK3β pathway signaling, and exert adverse impacts on renal electrolyte balance and BP regulation has not been explored. It is also unclear whether MR blockers could potentially have protective effects against e-cigarette-induced BP elevation through the GSK3β signaling pathway.

In this study, we explored the adverse effects and mechanisms of e-cigarette vapor exposure with or without nicotine on renal MR action, electrolytic balance, and BP regulation in C57BL/6J mice. To this end, GSK3β signaling and pre-receptor regulation of MR were analyzed with and without the treatment of the MR receptor antagonist eplerenone in mouse renal CCD M1 cells and in the kidneys of mice following exposure to e-cigarette vapors.

METHODS

The authors declare that all data are available within supporting Supplemental Material, which also contains detailed methods and material sources.

Animals

Ten-week-old C57BL/6 males were purchased from the Jackson Laboratory. Mice were housed in a SPF room maintained on a 12:12-h light-dark cycle with free access to water and standard chow. All animal experiments were approved by the Institutional Animal Care and Use Committees of Charles R. Drew University and were performed in accordance with the recommendation of the American Veterinary Medical Association.

E-cigarette exposure of mice

The e-cigarette exposure system (EcigAero 3eCig mouse model) was used to provide e-cigarette vapor for inhalation (AutoMate Scientific, Inc. CA).^35,36 Mice were exposed to aerosolized PBS vapor, aerosol nicotine-free or nicotine-containing e-cigarettes vapors for 6 weeks. We used actual e-cigarettes for exposure, consisting of a 50% propylene glycol (PG) and 50% vegetable glycerin (VG) base, with either 0% nicotine (0% e-cigarette) or 2.4% nicotine (2.4% e-cigarette), purchased from the website (https://vaporvapes.com). Concurrently, mice were randomly assigned to receive either solvent vehicle or the MR antagonist eplerenone (Sigma-Aldrich, St Louis, MO) (50 mg/kg/day, p.o.) by oral gavage,³⁷ starting at the first week until the end of the 6-week e-cigarette exposure.

Statistical analysis

All values are expressed as the mean ± SEM. Data distribution and comparisons between two groups were performed by unpaired Student t-test. To compare multiple groups, a two-way analysis of variance was used. The post hoc Tukey test was performed to reveal significant differences between groups. P<0.05 was considered statistically significant.

RESULTS

E-cigarette vapor inhalation triggers BP elevation and increases renal MR gene expression

To establish a causal relationship between e-cigarette aerosol inhalation and blood pressure (BP) elevation, we tested the impact of exposure to e-cigarette vapors with (E-Cig Nic) or without nicotine (E-Cig Veh) on BP, metabolic parameters, and renal MR. The mean systolic BP (SBP) was increased slightly at the first and second weeks and was markedly elevated at the third and fourth weeks and was elevated even further at the fifth and sixth weeks of exposure to E-Cig Nic (Figure 1A). However, no statistically significant effect was observed on diastolic BP (DBP) (Figure 1B) or heart rate (HR) (Figure 1C), although a trend towards increased diastolic BP was noted compared with the aerosolized PBS control. Additionally, mice exposed to E-Cig Nic also showed a significant increase in mean arterial pressure in mice (128±12.7 mm Hg) compared with PBS control mice (93± 8.6 mm Hg). In contrast, exposure to E-Cig Vehicle did not result in any significant changes in SBP, DBP, or HR compared with PBS controls (Figure 1A–1C). The average plasma nicotine levels in mice after E-Cig Nic exposure was 31.2 ± 7.6 ng/mL, which is similar to that in habitual cigarette smokers or E-Cig users^38,39 (Figure 1D). In parallel with the rise in SBP, E-Cig Nic exposure raised plasma corticosterone levels and lowered renin concentrations, without affecting aldosterone levels, compared to PBS controls (Figures 1E and 1F). Conversely, the levels of MR mRNA in the kidneys of mice on E-Cig Nic exposure were markedly increased compared with PBS controls (Figure 2A). Similarly, renal MR protein expression was increased by 1.6-fold and its nuclear protein content was elevated by 2.1-fold in E-Cig Nic-treated mice (Figure 2B and 2C). Double Immunofluorescence staining confirmed that exposure of E-Cig Nic vapor upregulated MR protein expression in the distal nephron using the distal tubular marker Peanut Agglutinin (Figures 2E and 2F). Moreover, the change in renal MR protein expression was positively correlated with SBP in E-Cig Nic-treated mice (Figure 2D). However, compared with PBS controls, E-Cig Veh exposure did not alter plasma corticosterone, aldosterone, and renin levels or renal MR abundance (Figures 1 and 2).

Figure 1. Exposure to e-cigarette vapor increases systolic blood pressure (SBP) and alters metabolic blood parameters in mice.

Mice were exposed to aerosolized PBS (PBS), nicotine-free (0 mg/mL) e-cigarette vapor, nicotine-containing e-cigarette vapor (24 mg/mL), or e-cigarette vapor with eplerenone treatment for 6 weeks. A. Systolic blood pressure (SBP). The time course of SBP (left) and the value at the end of E-Cig exposure (right). B. Diastolic BP after 6 weeks of exposure. C. Heart rate (HR). D. Plasma nicotine. E. Plasma corticosterone. F. Plasma aldosterone (left) and plasma renin levels (right). The e-cigarette vehicle without nicotine is denoted as (E-Veh); with 24 mg/mL nicotine as (E-Nic); and with 24 mg/mL nicotine and eplerenone as (E-Nic + Epl). Data are presented as mean ± SEM (n=7–8 mice per group). *P < 0.05 and **P < 0.01 vs. aerosol PBS- or E-Veh-treated mice.

Figure 2. Inhaling e-cigarette vapor elevates renal MR expression in mice.

A. The relative expression levels of MR mRNA were measured and normalized to 18S (n=6–8). B and C. Representative Western blot images from two animals per group were shown for total and nuclear MR. Each analyte was run on two independent blots, and final quantification was performed using samples from six animals per group (n=6). D. Correlation analysis between renal MR expression and systolic blood pressure (SBP). E. MR protein expression in renal distal tubules was assessed by double immunofluorescence staining using the distal tubular marker PNA. The white bar indicates 20 μm. F. MR fluorescence intensity was quantified from three non-overlapping fields per section per mouse using Fiji software (n=6). Data are presented as mean ± SEM. *P < 0.01 vs. aerosol PBS- or E-Veh-treated mice. *P<0.01 vs. aerosol PBS- or E-Veh-treated mice.

E-Cig vapor exposure enhances renal ENaC expression and disturbs electrolyte homeostasis

We next analyzed if exposure to E-Cig vapors impacts the expression of the renal MR target gene epithelial sodium channel (ENaC), a key regulator of renal sodium balance. In line with MR expression, the abundance of renal ENaCα mRNA and protein, along with their cleaved forms, was markedly increased by E-Cig Nic exposure compared with PBS controls (Figure 3A–3B). However, renal ENaCγ mRNA and protein expression showed no obvious change; but the levels of its cleaved protein were elevated in mice exposed to E-Cig Nic, compared with PBS controls (Figure 3A–3B). In contrast, E-Cig Nic exposure reduced urinary Na⁺ content and increased plasma Na⁺ levels with a reduced urinary Na⁺/K⁺ ratio compared with PBS controls (Figure 3C–3E). Furthermore, E-Cig Nic exposure increased urinary K⁺ levels and slightly decreased plasma K⁺ levels without affecting the urinary albumin/creatinine ratio (Figure 3C–3F). However, E-Cig Veh exposure did not alter renal ENaCα and ENaCγ expression nor the plasma Na⁺ and K⁺ and urinary Na⁺ levels.

Figure 3. Inhaling e-cigarette vapor stimulates renal ENaC and disrupts electrolyte balance in mice.

A. Renal ENaCα and ENaCγ mRNA levels were quantified by real-time RT-PCR and normalized to 18S. B. Representative Western blot images from two animals per group were shown for full-length and cleaved ENaCα and ENaCγ. Each analyte was run on two independent blots, and final quantification was performed using samples from six animals per group (n=6). C and D. Urinary Na⁺ and K⁺ levels were measured by flame photometry using 24-hour urine samples (C), along with the urinary Na⁺/K⁺ ratio (D). E. Plasma Na⁺ and K⁺ levels. F. The urine albumin /creatinine ratio was measured using assay kits (n=6). Data are presented as mean ± SEM. *P < 0.05 and **P < 0.01 vs. aerosol PBS or E-Veh-treated mice

E-Cig exposure decreases renal 11β-HSD2 expression and elevates the urinary corticosterone /11-dehydrocorticosterone ratio

Since 11β-HSD2 is known to protect MR signaling through the inactivation of GCs, the effects of E-Cig vapor exposure on renal 11β-HSD2 were examined. Immunofluorescence staining analysis showed that 11β-HSD2 expression was downregulated by E-Cig Nic exposure in the distal nephron (Figure 4A and 4B) and was negatively correlated with upregulated MR expression (Figure 2E). Real-time RT-PCR analysis showed that renal 11β-HSD2 mRNA levels were markedly decreased in mice on E-Cig Nic compared with PBS controls (Figure 4C). Similarly, renal 11β-HSD2 protein levels were decreased by 2.2-fold with the reduction of 11β-HSD2 activity by 43% in the kidneys of mice on E-Cig Nic compared with PBS controls (Figure 4D and 4E). In parallel with the changes in 11β-HSD2 expression, C/EBPβ mRNA and protein expression in the kidneys, the key transcriptional factor of 11β-HSD2, were markedly decreased by E-Cig Nic exposure compared with PBS controls (Figure 5D–5F). Conversely, the urinary corticosterone to 11-DHC ratio was increased by 6.3-fold in E-Cig Nic-exposed mice compared to controls (Figure 4F, P <0.01). However, there were no significant changes in the expression and activity of renal 11β-HSD2, urinary corticosterone/11-DHC ratio, or renal C/EBPβ abundance in E-Cig Veh-treated mice compared to PBS controls (Figures 4 and 5).

Figure 4. Inhalation of e-cigarette vapor containing nicotine decreases renal 11β-HSD2 and elevates the urinary corticosterone/11-dehydrocorticosterone (11-DHC) ratio in mice.

A. Double immunofluorescence staining was used to analyze 11β-HSD2 protein expression in the distal tubules of kidney sections using the distal tubular marker PNA. The white bar indicates 20 μm. B. 11β-HSD2 fluorescence intensity was quantified from three non-overlapping fields per section per mouse using Fiji software (n=6). C. Relative expression of renal 11β-HSD2 mRNA levels was normalized to 18S (n=8). D. A representative Western blot image from two animals per group was shown for 11β-HSD2. Each analyte was run on two independent blots, and final quantification was performed from six animals per group (n=6). E. 11β-HSD2 activity is expressed as the percentage conversion of [³H] corticosterone to [³H]11-DHC. F. The ratio of corticosterone to 11-DHC in 24-hour urine samples from each group of mice (n=6). Data are presented as mean ± SEM. *P < 0.01 vs. aerosol PBS or E-Veh-treated mice.

Figure 5. Eplerenone suppresses renal GSK3β signaling induced by inhaling e-cigarettes containing nicotine in mice.

A and D. Real-time RT-PCR analysis was conducted to measure renal GSK3β and C/EBPβ levels (n=6). B, E, and F. Representative Western blot images from two animals per group were shown for GSK3β, pSer⁹GSK3β, C/EBPβ, and pSer¹⁰⁵C/EBPβ. Each analyte was run on two independent blots, and final quantification was performed from six animals per group (n=6). C. GSK3β activity was determined using ATP as a substrate and is expressed as the percentage conversion of ATP to ADP (n=6). Data are presented as mean ± SEM. *P < 0.01 vs. aerosol PBS- or E-Veh-exposed mice.

MR blocker eplerenone blunted the progression of BP elevation induced by E-Cig exposure by modulating MR pre-receptor signaling through inhibition of pSer⁹GSK3β activation

As shown in Figure 1A, eplerenone treatment significantly decreased the elevated mean SBP levels in mice exposed to E-Cig Nic and normalized the E-Cig Nic-induced decreases in renin levels, although it did not affect elevated plasma corticosterone or aldosterone levels (Figure 1E–1F). In contrast, eplerenone restored urinary Na⁺ and plasma Na⁺ levels to those of PBS controls after E-Cig Nic exposure (Figure 3C and 3E). Moreover, eplerenone mitigated the elevated urinary K⁺ levels and reduced Na⁺/K⁺ ratio caused by E-Cig Nic, though plasma K⁺ and albumin/Cre levels remained unchanged (Figure 3C–3F). Furthermore, eplerenone reversed the increased ENaCα abundance and reduced the elevated cleaved protein content of renal ENaCα and ENaCγ with reduction of renal MR expression, while maintaining ENaCγ abundance (Figure 3A–3B and Figure 2A–2C). Consistent with changes in MR expression, the renal pSer⁹GSK3β content and activity⁴⁰ were markedly increased after E-Cig Nic exposure without changes in GSK3β mRNA and total protein expression, and were normalized by eplerenone treatment (Figure 5A–5C). In addition, eplerenone treatment also ameliorated the decreased C/EBPβ (Figure 5D–5F) blunted the reduction in the expression and activity of 11β-HSD2 within the kidneys, and reversed the elevated urinary corticosterone/11-DHC ratio in mice exposed to E-Cig Nic (Figure 4).

E-Cig aerosols-mediated activation of MR via downregulation of 11β-HSD2 in CCD M1 Cells

To confirm the relevance of the in vivo findings, we tested the direct impact of E-Cig aerosols in mouse CCD M1 cells. As shown in Figure S1A, exposing CCD M1 cells to diluted concentrations (0.05 −0.4 puffs/mL)^41,42 of E-Cig aerosol Nic^43,44 for 24h resulted in a dose-dependent increase in MR mRNA and protein expression levels compared with control cells. Conversely, CCD M1 cells exposed to aerosol from E-Cig Nic vapor for 24 h exhibited reduced 11β-HSD2 mRNA and protein levels compared with controls (Figure S1B–S1C). However, treating CCD M1 cells with E-Cig vehicle aerosol (0% nicotine) for 24 h did not affect MR and 11β-HSD2 expression in these cells (Figure S1A–S1C).

We next examined if enhancing 11β-HSD2 might modulate the effects of E-Cig Nic aerosols on MR expression. As shown in Figures S1D–S1F, 11β-HSD2 mRNA and protein levels were markedly increased in CCD M1 cells transfected with 11β-HSD2 plasmid cDNA compared with cells transfected with control plasmid. In contrast, overexpression of 11β-HSD2 in cells attenuated the E-Cig aerosol Nic -mediated increases in MR mRNA and protein expression levels, along with decreasing the nuclear MR content (Figure S1D–S1F), indicating that E-Cig aerosol nicotine-induced MR expression is mediated in part by reduction of 11β-HSD2. In addition, elevating 11β-HSD2 through overexpression exerted effects comparable to those of eplerenone treatment in reducing E-Cig aerosol nicotine-induced changes in MR and 11β-HSD2 expression in these intact cells (data not shown). Additionally, overexpression of 11β-HSD2 mitigates the E-Cig aerosol Nic-mediated suppression of pSer¹⁰⁵C/EBPβ and partially reverses the increase in pSer⁹GSK3β protein expression induced by E-Cig aerosol Nic in these cells (Figure S1G and S1H).

E-Cig aerosols modulation of MR and 11β-HSD2 expression via activation of pSer⁹GSK3β signaling pathway

Because E-Cig with nicotine exerts its adverse effects through activation of pSer⁹GSK3β, we tested whether inhibition of GSK3β kinase could modulate the E-Cig aerosol-induction of MR. Western blot analysis revealed that CCD M1 cells exposed to a concentration of 0.1 puffs/mL of E-Cig aerosol Nic, yielding an average nicotine amount of 2.37 μM (a dose comparable to that seen in habitual smokers)^38,45 showed increased pSer⁹GSK3β content and elevated MR abundance and nuclear content compared with controls (Figure 6A–6D). In contrast, treating cells with 0.1 puffs/mL of E-Cig aerosol Nic in the presence of the GSK3β inhibitor SB216763 (1μM) did not stimulate MR gene expression with inactivation of pSer⁹GSK3β compared to vehicle-treated cells (Figure 6A–6D), suggesting that activation of GSK3β is required for the E-Cig aerosol-mediated activation of MR.

Figure 6. The effects of GSK3β inhibitor in CCD M1 cells.

Cells were treated with nicotine-containing (24 mg/mL) e-cigarette aerosol (0.1 puffs/mL; E-Nic) in the absence or presence of the GSK3β inhibitor SB 216763 (10⁻⁶ mol/L; SB) for 24 h. A. The relative expression levels of GSK3β, MR, and C/EBPβ mRNA were normalized to 18S. B-D. Western blot analysis showed the protein expression levels of total GSK3β, phosphorylated Ser⁹GSK3β (pSer⁹GSK3β) (B), total MR (C), and nuclear MR (D) in these cells treated with e-cigarette aerosol in the absence or presence of GSK3β inhibitor SB 216763 for 24 h. E-F. Inhibition of Ser⁹GSK3β by its inhibitor reduced the effects of aerosol nicotine on pSer¹⁰⁵C/EBPβ and 11β-HSD2 expression in these cells. Data represent the mean ± SEM from three to four independent experiments. *P < 0.05 and **P < 0.01 vs. controls.

Since GSK3β has emerged as a negative regulator of C/EBPβ, we examined if inhibition of GSK3β could affect C/EBPβ and 11β-HSD2. Coincubation of cells with both GSK3β inhibitor and E-Cig Nic aerosol blunted E-Cig aerosol Nic-induced suppression of C/EBPβ mRNA expression and pSer¹⁰⁵C/EBPβ content (Figure 6A and 6E). Moreover, the reduction of 11β-HSD2 protein expression induced by E-Cig aerosol Nic was attenuated by incubation with the GSK3β inhibitor in these cells (Figure 6F). However, the GSK3β inhibitor alone (1μM) did not affect C/EBPβ and had no impact on 11β-HSD2 in these cells compared with controls (Figure 6A, 6E, and 6F).

Nicotinic receptor mediates E-Cig vapors modulation of GSK3β signaling and MR activation

To further verify whether the harmful effects of E-Cig exposure on renal cells occur in a nicotine-dependent manner, cells were exposed to E-Cig aerosols in the presence or absence of the nicotine antagonist hexamethonium (Hex). As expected, treating CCD M1 cells with Hex (1μM) abolished the E-Cig aerosol Nic-induced activation of pSer⁹GSK3β and reduced MR expression and its nuclear levels (Figure S2A–S2D). Similarly, the reductions in C/EBPβ and 11β-HSD2 expression induced by E-Cig aerosol Nic were reversed by Hex treatment in these cells (Figure S2E–S2F). However, Hex treatment had no significant effects on GSK3β or C/EBPβ expression, and it did not affect 11β-HSD2 and MR protein levels in comparison with control cells (Figure S2).

Discussion

Emerging evidence indicates that e-cigarette smoking increases the predisposition to hypertension, but the underlying mechanisms remain largely unknown. We present here data for the first time showing that chronic e-cigarette vapor exposure induces activation of renal MR signaling and increases mean arterial pressure in mice. We found that mice exposed to nicotine-containing e-cigarettes exhibited increased renal MR expression and elevated systolic blood pressure (SBP) in response to elevated plasma nicotine levels. E-cigarette exposure also increased renal cleaved ENaCα and ENaCγ content, major markers of ENaC activity that were activated by MR, accompanied by elevated plasma sodium and a decreased urinary Na⁺/K⁺ ratio corresponding with BP elevation. Increased abundance of renal MR expression by nicotine e-cigarettes could stimulate its target gene, ENaC expression, which would directly promote aberrant urinary Na⁺ reabsorption and decrease natriuresis. This could result in Na⁺ retention and thus be linked to the development of hypertension. This interpretation is supported by recent reports revealing that nicotine reduces urinary Na⁺ extraction and increases plasma Na⁺ levels in both rats and cigarette smokers.^46,47 Furthermore, we demonstrated that plasma renin levels decreased in response to the induction of renal ENaC activity and Na⁺ retention, which corresponded with elevated BP in mice exposed to e-cigarette aerosols. This is consistent with renin secretion is down-regulated by renal ENaC activity and BP.⁴⁸ To our knowledge, MR signaling and electrolyte balance have not been measured in rodents or humans following e-cigarette exposure. Our findings suggest that BP elevation induced by e-cigarettes may arise, in part, from enhanced MR signaling in the kidneys. In addition, e-cigarette smoking could also affect peripheral sympathetic activity, cardiovascular system, renal function, and immune fibrosis,^10,49 all of which are linked to the pathogenesis of hypertension.

It has been reported that e-cigarette exposure increases BP, primarily due to aerosol nicotine inhalation rather than non-nicotine constituents in e-cigarettes.^8,50 We observed that e-cigarette exposure induced renal MR signaling and an increase in BP, both of which are correlated with elevated plasma nicotine levels similar to those found in habitual smokers and e-cigarette users.^38,39 In contrast, inhaling e-cigarettes without nicotine causes no changes in renal MR signaling or BP response. Our findings suggest that the adverse effects of e-cigarettes on renal MR signaling and BP regulation may be primarily attributed to inhaling nicotine. This is in agreement with recent reports that nicotine stimulates renal MR expression in mice,⁵¹ and this study is the first to find a similar response from inhaling nicotine from e-cigarette. Endorsing this concept, our cell culture findings indicate that the upregulation of MR expression in CCD M1 cells by e-cigarette aerosols is specifically due to aerosolized nicotine, and not to the vehicle aerosols. Conversely, we observed that the nAChR antagonist Hex effectively blocked the activation of MR signaling induced by nicotine aerosols in these cells. Our data provide the first evidence that nicotine aerosols directly exert adverse effects on CCD cells and MR signaling. Taken together, these findings suggest that nicotine aerosols enhance renal MR signaling through a nicotine receptor-mediated mechanism. This is in line with the notion that aerosol nicotine delivered by e-cigarettes has similar detrimental effects on renal cells as those of traditional smoking, which disrupts renal function through binding to nAChRs.^50,52,53 To our knowledge, the modulation of MR by nicotine, including that from e-cigarettes, via nAChR signaling, has not yet been explored in either human or animal models.

Normal MR activity is known to be protected by 11β-HSD2 inactivating GCs in target tissues. In line with this concept, we observed a positive correlation between increased renal MR activation and decreased renal 11β-HSD2 expression and activity in mice exposed to e-cigarettes. Double immunofluorescence staining reveals a downregulation of 11β-HSD2 accompanied by an upregulation of MR expression in the renal distal tubules of mice exposed to e-cigarette aerosols. These data indicate that inhaling nicotine e-cigarettes impairs the conversion of corticosterone to 11-DHC in the kidneys of mice. This was validated using renal CCD cells, where e-cigarette nicotine aerosols downregulated 11β-HSD2 and stimulated MR expression, unlike cells treated with vehicle aerosols. Given that MRs are inherently nonselective and can be activated by both aldosterone and GCs,^17–20 a reduction in renal 11β-HSD2 by e-cigarettes could lead to higher occupation of MR by elevated local GC levels in the distal nephron. This aligns with reports that nicotine elevates plasma GC levels and decreases 11β-HSD2 expression in both rat placentas and mouse kidneys.^51,54 However, to date, no studies have been published investigating the effects of e-cigarette aerosols on the pre-receptor metabolism of GCs. Conversely, inducing overexpression of 11β-HSD2 attenuated the nicotine aerosol-mediated induction of MR expression in CCD M1 cells. Our data suggest that the reduction of renal 11β-HSD2 may be responsible for the e-cigarette-mediated activation of renal MR signaling.

A possible mechanism that may play a substantial role in mediating the adverse effects of inhaling nicotine from e-cigarettes on renal 11β-HSD2 and MR signaling is the GSK3β/C/EBPβ pathway. Indeed, GSK3β has been shown to be a negative regulator of C/EBPβ,^31,32 which is a key transcription activator of 11β-HSD2.⁵⁵ Consistent with this notion, we found that the levels of pSer⁹GSK3β increased in response to suppression of C/EBPβ, with a concomitant reduction in 11β-HSD2 in the kidneys of mice exposed to e-cigarettes. This aligns with findings that nicotine increases GSK3β in mouse brain tissue⁵⁶ and decreases C/EBPβ in the airway epithelium of smokers.³⁴ This suggests a potential mechanism whereby aerosol nicotine induces pSer⁹GSK3β, thereby reducing C/EBPβ and leading to the suppression of 11β-HSD2. Moreover, induction of MR expression by e-cigarettes was positively correlated with elevated pSer⁹GSK3β levels in the kidneys, suggesting that activation of GSK3β itself could promote renal MR gene expression. Induction of GSK3β could enhance corticosterone-activated MR gene transcription, thereby contributing to enhanced MR expression. This is in line with the finding that activation of GSK3β signaling itself leads to excess steroid-induced MR expression in renal cells.³⁰ Moreover, GSK3β is activated by GCs and nicotine,^56–59 and elevated plasma levels of nicotine and corticosterone may help explain the elevated renal pSer⁹GSK3β observed in mice on e-cigarettes. In contrast, inhibition of pSer⁹GSK3β with its inhibitor diminished the MR expression induced by nicotine aerosols, while simultaneously stimulating C/EBPβ and 11β-HSD2 in CCD M1 cells. These findings suggest that the activation of GSK3β signaling may trigger aerosol nicotine-induced MR expression and reduction of 11β-HSD2, consistent with GSK3β being a key transcriptional mediator of ligand-dependent gene transcription. To our knowledge, this study is the first to demonstrate the impact of e-cigarette vapors on the GSK3β/C/EBPβ pathway.

MR blockers are known to prevent steroid-induced activation of MR and subsequent hypertension.⁶⁰ In the current study, we observed that treatment with the MR blocker eplerenone reduced the e-cigarette-induced activation of renal MR signaling by GCs and effectively attenuated the associated BP elevation. Moreover, eplerenone reduced the renal levels of cleaved ENaCα and ENaCγ proteins, normalized elevated plasma Na⁺ levels, and concurrently reversed abnormal urinary Na⁺ excretion, and corrected low renin levels in mice exposed to e-cigarettes. These data provide the first evidence that eplerenone may exert beneficial effects in protecting against the adverse effects of e-cigarette aerosols on renal electrolyte imbalances and BP elevation by blocking excessive MR occupancy in the kidneys. This aligns with the notion that MR blockers can inhibit MR activation-induced Na⁺ retention and hypertension through a reduction in ENaC abundance.^51,61

Although the MR antagonist spironolactone may inhibit adrenal secretion of GCs,⁶² which could contribute to reduced MR activation and hypertension, treatment with eplerenone did not modulate the elevated plasma corticosterone levels in mice exposed to e-cigarettes. Such effects would be consistent with previous reports that eplerenone does not interfere with the biosynthesis of adrenocortical steroids.⁶³ In contrast, we observed that the reduction of renal MR and BP by eplerenone correlates with the reversal of the e-cigarette-induced high urinary corticosterone/11-DHC ratio. These data suggest that eplerenone modulates the ability of renal 11β-HSD2 to deactivate corticosterone into 11-DHC, thus attenuating local elevated GCs of occupancy of MR and the resultant BP elevation, regardless of circulating GC concentrations. Conversely, the ratio of urinary corticosterone to 11-DHC was markedly increased in response to the activation of renal MR signaling and BP elevation in mice exposed to nicotine e-cigarettes. Our data support the possibility that a reduction in the urinary corticosterone/11-DHC ratio may serve as a potential metabolic signal or biomarker for the eplerenone blockade of renal MR activation and subsequent elevation in BP. In addition, eplerenone also inhibited the e-cigarette-induced pSer⁹GSK3β and upregulated C/EBPβ, which correlated with a reduction in MR expression and a stimulation of 11β-HSD2 in the kidneys. These results indicate that the beneficial effects of eplerenone against e-cigarette-induced BP elevation occur partly through the reduction of GSK3β activation. This agrees with the notion that spironolactone protects renal function through inhibition of GSK3β overactivity.⁵¹ Moreover, inactivation of pSer⁹GSK3β by its inhibitor showed effects comparable to those of eplerenone in attenuating nicotine aerosol-induced pSer⁹GSK3β content and rescuing C/EBPβ and 11β-HSD2 expression in CCD cells. Inhibiting pSer⁹GSK3β can stimulate C/EBPβ, which promotes 11β-HSD2 gene expression by enhancing its transcription, thus limiting GC occupancy of renal MR and subsequently reducing BP. These data support our hypothesis that modulation of GSK3β signaling may be an important additional mechanism that accounts for eplerenone attenuating e-cigarette-induced BP elevation. These results are the first to demonstrate a role for eplerenone in alleviating the adverse impacts of e-cigarettes on renal health and BP raise through the alteration of GSK3β signaling.

In conclusion, our results reveal that e-cigarette exposure has profound effects on renal electrolytic disorder and increased BP primarily by triggering activation of renal MR signaling with repression of 11β-HSD2. Our results also indicate that nicotine aerosols from vapors are likely to be the key component that elicits this adverse response. Furthermore, eplerenone seems to exert its beneficial effects on reducing e-cigarette-associated BP elevation not only through the blockage of MRs but also partly through the alteration of GSK3β signaling in the kidneys.

Perspectives

Our studies demonstrate that inhaling e-cigarettes with nicotine adversely impacts renal electrolyte balance and BP elevation by triggering the activation of renal MR signaling due to disturbed intra-renal inactivation of glucocorticoids. Our results also illustrate that inhaling aerosolized nicotine from e-cigarette vapor is likely the key component eliciting these adverse responses. Eplerenone potentially has a beneficial effect in combating the adverse effects on renal metabolism and BP elevation caused by nicotine inhalation. Future studies are required to clarify the long-term impacts of e-cigarette use on renal health and BP regulation, and to validate the relationship between BP and electronic smoking.

Supplementary Material

Supplemental Materials

Expanded Methods

References: 35, 36, 40, 41,42, 43, 44, 51.

Figures S1–S2

Novelty and Relevance.

What Is New?

We discovered that aerosol nicotine inhalation-induced renal electrolyte disorders and BP elevation from e-cigarette vapor are associated with increased activation of renal MR signaling, and these effects are prevented by eplerenone. In contrast, e-cigarettes without nicotine elicited no response in MR signaling effects. We also identified that GSK3β signaling may be responsible for the e-cigarette-induced alterations in MR expression, through modulation of glucocorticoid inactivation via a nicotine receptor-mediated mechanism in kidneys. These findings show that the enhancement of renal MR signaling by aerosol nicotine may represent a novel mechanism accounting for the risk of electronic smoking-induced hypertension.

What Is Relevant?

E-cigarette use increases the predisposition to hypertension in users, but the potential impact of renal MR signaling underlying the BP elevation induced by electronic smoking remains unknown. Normal renal MR signaling is crucial for regulating electrolyte balance and BP control. Here, we provide novel evidence that inhaling aerosol nicotine from e-cigarettes enhances GSK3β signaling, which is required for ligand-inducible MR expression and associated with reduced the pre-receptor inactivation of glucocorticoids within the kidneys, thus disturbing renal electrolytic balance and subsequently elevating blood pressure.

Clinical/Pathophysiological Implications

Our results reveal that e-cigarette smoking has profound effects on renal electrolytic disorder and BP elevation by triggering activation of renal MR signaling. Our results also illustrate that nicotine aerosols from e-cigarette vapor are likely to be the key component that elicits this adverse response. Furthermore, the MR antagonist eplerenone appears to exert its beneficial effects on reducing BP elevation associated with e-cigarette smoking not only through the blockade of MRs but also partly by altering GSK3β signaling in the mouse kidneys. These findings suggest that modulation of renal MR signaling could offer a potential therapeutic approach for treating hypertension and other cardiovascular disorders mediated by electronic smoking in users.

Nonstandard Abbreviations and Acronyms:

E-Cig

Electronic Cigarette

E-Cig Nic

E-cigarettes with nicotine

E-Cig Veh

E-cigarettes without nicotine

MR

Mineralocorticoid Receptor

GSK3β

Glycogen Synthase Kinase-3β

C/EBPβ

CCAAT/enhancer-binding protein-β

11β-HSD2

11β-hydroxysteroid dehydrogenase type 2

SBP

Systolic Blood Pressure

DBP

Diastolic Blood Pressure