Renal Function in Male Sprague-Dawley Rats Concurrently Exposed to Long-Term Nicotine (3-{1-Methyl-2-Pyrrolidinyl}Pyridine) and Methylated Spirits (Methyl Alcohol)

Abstract

Abuse of nicotine and methylated spirits is a global problem. The current study determined the concurrent influence of nicotine and methylated spirits on renal hemodynamics. Two series of experimental protocols were designed: conscious and anesthetized vehicle loading. Conscious animals received nicotine (0.1 mg.kg⁻¹ bwt, 0.26–0.30 mL), methylated spirits (1.0 g.kg ⁻¹ bwt, 0.26–0.30 mL), combined nicotine and methylated spirits, and control animals (water, 0.26–0.30 mL). Anesthetized animals were challenged with a continuous jugular infusion of 0.077M NaCl. Plasma nicotine concentration was significantly elevated in combined conscious treatments by comparison with animals infused nicotine alone. Plasma arginine vasopressin was significantly attenuated in combined conscious groups, and those infused methylated spirits alone. Aldosterone was elevated in all conscious groups. Both plasma ethanol and methanol concentrations were elevated in rats concurrently administered nicotine and methylated spirits compared with those given methylated spirits alone. Urinary Na⁺ levels were significantly elevated in all anesthetized groups associated with attenuated aldosterone concentrations. Plasma nicotine concentrations were increased in combined treatments. Plasma ethanol levels were significantly reduced and elevated in rats concurrently exposed to nicotine and methylated spirits, respectively. The present study suggests that chronic exposure to methylated spirits alone and in combination with nicotine increases urinary Na⁺ loss. The renal toxicity is manifested hypothetically via elevations in plasma nicotine and methanol concentrations. This implies that people who concurrently consume methylated spirits and smoke cigarettes have an increased risk of renal failure by being predisposed to fluid and electrolyte disturbances.

Keywords:

• kidney
• methanol
• methylated spirits
• nicotine
• rat model

INTRODUCTION

To date, there are no studies published in recognized, impact factor-classified journals considering the concurrent exposure of nicotine and methylated spirits on renal function. Industrial methylated spirits is classically a combination of 5% (v/v) methanol and 95% (v/v) ethanol.[1] A safe daily dose of methanol (methyl alcohol; Mr 32.04 g/mol) in an adult is 2 g, whereas doses of 8 g and excess are toxic. In 2001, the EU general limit for naturally occurring methanol of 10 g methanol per litre of ethanol equates to 0.4% (v/v) methanol at 40% ethanol.[1] The current workplace exposure limits include: Health and Safety Executive (HSE) Workplace Exposure Limit (WEL): 8-h Time-Weighted Average (TWA), 266 mg.m⁻³ and 200 ppm; 15-min Short-Term Exposure Limit (STEL): 333 mg.m⁻³ and 250 ppm.[2] In Australia, methanol poisoning is rare due to legislated removal of methanol from methylated spirits.[3] However, methylated spirits are commonly consumed globally as a cheap alternative to liquor. The strongest predictor of death or a poor outcome following methanol ingestion was a pH < 7.0.[4] Methanol metabolites, formic acid, and formaldehyde, produced by alcohol dehydrogenase in the liver, contribute to histo-pathological degeneration of the optic nerve and irreversible blindness.[5] Incidents of diffuse encephalopathy with residual pseudobulbar palsy and dementia have been recorded.[6] Health effects include headache, dizziness, dermatitis, and conjunctivitis.

The current study extends investigations on renal function following concurrent administration of nicotine and ethanol[7] and chloroquine and ethanol[8–10] on renal function in male Sprague-Dawley rats. The effect of tobacco usage on renal function has been reviewed.[11] The aim therefore was to determine in a laboratory study the concurrent influence of nicotine and methylated spirits on renal hemodynamics.

MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats (265–320 g bwt) were bred and housed at 24°C in the Animal House attached to the Faculty of Medicine and allowed free access to food (Mouse Comproids, National Foods, Harare, Zimbabwe) and water ad libitum. Licensing to perform vivisections was granted by the Ministry of Agriculture via the Scientific Animal Experiment Act, 1963.

Renal Function Studies

Series 1: Four-Weekly Influence of Nicotine and/or Methylated Spirits

All animals were maintained on a 12h light/ 12h dark regime with ample access to food and water, and housed in separate metabolism cages (NKP cages, Dartford, Kent, UK), which were disinfected daily. Rats were then grouped and administered every second day for 4 wk with the following:

• nicotine: (3-{1-methyl-2-pyrrolidinyl}pyridine), Sigma, St. Louis, Missouri, USA; 0.1 mg.kg⁻¹ bwt, 0.26–0.30 mL,

• methylated spirits: Datlabs (Pvt.) Ltd., methyl alcohol, 1.0 g.kg ⁻¹ bwt, 0.26–0.30 mL),

• combined nicotine (0.1 mg.kg⁻¹ bwt) and methylated spirits (1.0 g.kg ⁻¹ bwt), and

• control animals (water, 0.26–0.30 mL).

Administration by gavage employed a bulbed steel tube passed orally into the stomach. Daily weight and volume measurements of food and water consumed respectively were recorded. Body weight was monitored on a daily basis. Urine volume and total urinary outputs of Na⁺ and K⁺ were determined from 24-h samples.

Series 2: Hypotonic Saline Challenged Rats Acutely Infused Nicotine and/or Methylated Spirits

Following 4-wk treatment, detailed renal function studies were conducted 24-hr after the last treatment by inducing anaesthesia via an intraperitoneal injection of trapanal [sodium 5-ethyl-5’-(1-methyl-butyl)-2-thio-barbiturate, Byk Gulden, Konstanz, Germany] in the right ventral abdomen at 0.11 g.kg⁻¹ bwt. The right jugular vein was cannulated with polyethylene tubing (internal diameter, i.d., 0.86 mm; external diameter, o.d., 1.27 mm, Clay Adams, New Jersey, USA) to allow intravenous infusion of 0.077 M NaCl. The left carotid artery was also cannulated with polythene tubing (i.d., 0.58 mm; o.d., 0.96 mm, Clay Adams) and then connected to a transducer (Grass Polygraph, Model 790, Grass Instruments, Quincy, Massachusetts, USA) for blood pressure measurements. The urinary bladder was cannulated with polyethylene tubing of the same size via an abdominal incision. Each rat was tracheotomized to maintain a clear airway. The body temperature was maintained with a heated table. Rats were placed on a continuous infusion of 0.077 M NaCl at 150 μL.min⁻¹ (Sage Syringe Pump Model 351) and a 3-h equilibration period was allowed. Following this, consecutive 20-min urine collections were made into pre-weighed plastic vials over the subsequent 4 h.

Analytical Methods

Measurement of Electrolytes

Urine volume was computed gravimetrically following collection in pre-weighed 5mL vials. Na⁺ and K⁺ were determined by Flame Photometry (Corning model 435 Flame Photometer, Corning Ltd., Halstead, UK).

Glomerular Filtration Rate (GFR) Measurements

GFR was measured using clearance of inulin in anesthetized groups of rats prepared for renal studies. Each rat was given a priming dose (0.3 μCi in 0.3 mL saline) of [³H] inulin (Amersham, Buckinghamshire, UK; specific activity 1.74 Ci mmol⁻¹) and then placed on a continuous intravenous infusion of 0.077 M NaCl containing inulin (0.14 μCi. min⁻¹) at 150 μL min⁻¹ throughout the experimental period. Blood samples (200 μL) were collected at 1h intervals into cooled heparinized tubes throughout the 4-h post-equilibration period for measurement of hematocrit prior to analysis of separated plasma. The radioactivity embedded in the aliquots of urine (10 μL) and plasma (10 μL) was counted on Minaxi β Tri-Carb 4000CA series liquid Scintillation Counter (Packard Instruments, Downer's Grove, Illinois, USA).

Plasma AVP and Aldosterone Measurements

Following decapitation of unanesthetised animals by a laboratory approved method,[12] blood was collected into cooled heparinized containers 24 h following completion of 4 wk treatment. Control animals were given an equal volume of water 24 h after the last treatment. Blood was also collected into pre-cooled heparinized containers from decapitated anesthetized rats prepared for renal studies after the 4 h post-equilibration period. Blood was spun at 3,000 rpm (800 g) (Sorvall RT6000 Refrigerated Centrifuge, Dupont Company, Newtown, Pennsylvania, USA) for 10 min at −4°C, and the plasma separated via pipette and was stored at - 20°C until measurement of aldosterone and AVP.

Plasma aldosterone was measured by Coat-A-Count using a kit purchased from Diagnostic Products, Los Angeles, California, USA. The principle of the test employed a solid-phase radioimmunoassay procedure based on aldosterone-specific antibodies immobilized to the walls of a polypropylene tube. The lower limit of detection was 44 fmol.L⁻¹. Inter- and intra-assay coefficients of variation were 8.1% (n  =  20) and 8.3% (n  =  20), respectively. AVP was extracted as described elsewhere[13] using the DSL-1800 Arginine Vasopressin Radioimmunoassay kit from Diagnostic Systems Laboratories, Webster, Texas, USA. Vasopressin was extracted from plasma using Sep Pak C18 cartridges (Millipore Water Associates, Harrow, Middlesex, UK). The lower limit of detection was 0.5 fmol.L⁻¹, and intra- and inter-assay coefficients of variations were 7.7% (n  =  12) and 11.9% (n  =  12), respectively.

Plasma Nicotine, Methanol, and Ethanol Measurements

Blood was collected into pre-cooled heparinized containers from chronically treated animals 24 h after the last administration, and from parallel groups of animals prepared for renal studies after completion of the 4-h post-equilibration period. Blood was centrifuged at 3,000 rpm (800 g; Sorvall RT6000 Refrigerated Centrifuge, Dupont Company, Newtown, USA) for 10 min at – 4°C, and plasma was separated by pipette and subsequently stored at –20°C. All parameters were measured 12 h following separation.

Plasma nicotine determinations were performed according to methods detailed elsewhere.[14],[15] The method employed high performance liquid chromatography in combination with mass spectrometric detection (Jasco UK, Great Dunmow, Essex, UK). The column consisted of phenyl Novapak with a mobile phase consisting of 50% 10 mM ammonium formate (pH 3.3) and acetonitrile (50:50, vol/vol) (Jasco UK, Great Dunmow, Essex, UK). The lower limits of detection, precision, and accuracy were determined using results from the quality control samples assayed with the experimental samples. The lower limit of detection was 5 ng.mL⁻¹. The precision of the controls was < 14.4% at low concentration (≤ 7.5 ng.ml⁻¹), 11.1% at medium concentration (≤ 75 ng.ml⁻¹), and 12.6% at high concentration (≤ 750 ng.ml⁻¹) of nicotine. The accuracy of the controls approximated −5.71% (low), −12.0% (medium) and −5.31% (high) concentrations of nicotine.[15]

Plasma methanol and ethanol concentrations were measured by gas chromatography (Philips PYE Unicam Series 304 Chromatograph, London, UK) equipped with a flame-ionization detector, a glass column (2 m  ×  22 mm) packed with 50% Porapak P and 50% Porapak Q. Selective absorption of volatile compounds was set at 98–103%. Nitrogen was used as a carrier gas to elute volatiles from the column. The column temperature was maintained at 150°C, the injection port at 210°C, and the detector at 270°C. The gas ratio injected into the burner was 2 hydrogen: 1 oxygen. A flame-ionization detector response was recorded using a HP 3396A integrator (Hewlett Packard, Avondale, Pennsylvania, USA). The internal standard used was 1-butanone. The intra- and inter-assay coefficients of variation were 5.08% (n  =  8) and 7.25% (n  =  8), respectively.

Statistical Analysis

Values were presented as means ± SEM. All data were analyzed following input into SPSS (Chicago, Illinois, USA) using ANOVA-1 (99% CI) and Scheffe's multiple comparison to resolve any apparent differences.

RESULTS

Series 1: Renal Effects—Chronic Indicator

Observations on the behavior of the rats based on work by Spangenberg et al.[16] provided no evidence that the animals were suffering from sickness or ill-health at the end of the 4 wk. period. Control rats steadily gained weight (see Table 1). Animals administered nicotine alone lost weight progressively, whereas other treatments saw weight gains. The latter recordings were in opposition to an attenuated food intake over the 4 wk period. Treatment with methylated spirits and combined nicotine/methylated spirits resulted in reduced urine flow in week 1. This attenuation was demonstrated throughout the 4 wk period in the former. Weekly Na⁺ loss was evident from week 3 onward, but was elevated throughout the 4 wk period in animals were treated with methylated spirits and combined nicotine/methylated spirits.

Table 1 Weekly determinations of body weight (% change) and masses of food consumed (g.day⁻¹), and weekly determinations of urine flow (mL.day⁻¹) and Na⁺ excretion (mmol.day⁻¹) rates in conscious control and animals treated every second consecutive day for 4 wk with nicotine and/or methylated spirits (n  =  8 in each group)

	Week
Treatment	1	2	3	4
Food intake
Control	26 ± 1	26 ± 1	27 ± 1	27 ± 1
Nicotine	21 ± 1	19 ± 1	17 ± 1	16 ± 1
M. spirits	19 ± 1	18 ± 2	18 ± 2	17 ± 2
Nicotine/M.spirits	20 ± 1	17 ± 1	18 ± 1	17 ± 1*
Body wt. change
Control	+4 ± 1	+5 ± 1	+7 ± 1	+9 ± 1
Nicotine	+2 ± 1	–1 ± 1	–5 ± 1*	–7 ± 1
Methylated spirits	+ 7 ± 1^†	+ 9 ± 1^†	+ 13 ± 1^†	+ 17 ± 1*^†
Nicotine/methylated spirits	+ 9 ± 1^†	+ 12 ± 1^†	+ 16 ± 1*^†	+ 18±1*^†
Urine flow
Control	8 ± 1	9 ± 1	9 ± 1	9 ± 1
Nicotine	9 ± 1	10 ± 1	10 ± 1	10 ± 1
Methylated spirits	5 ± 1*^†	6 ± 1*^†	4 ± 1^†	5 ± 1^†
Nicotine/methylated spirits	6 ± 1*^†	8 ± 1	9 ± 1	9 ± 1
Weekly Na^+
Control	14 ± 1	14 ± 2	15 ± 1	14 ± 2
Nicotine	12 ± 1	12 ± 1	10 ± 1	6 ± 1^†
Methylated spirits	17 ± 1^†	19 ± 1^†	21 ± 1*^†	23 ± 1^†
Nicotine/methylated spirits	19 ± 1^†	20 ± 1*^†	22 ± 1^†	25 ± 1^†

*p < 0.01 by comparison with control animals.

^†p < 0.01 by comparison with rats administered nicotine alone.

Plasma nicotine concentration was significantly elevated in combine treatment by comparison with animals infused nicotine alone (see Table 2). Plasma AVP was significantly attenuated in combined treatment groups and those infused methylated spirits alone. ALD was elevated in all groups. Although plasma Na⁺ was significantly elevated in all treatment groups, plasma K+ and pH did not differ significantly from controls. Both plasma ethanol and methanol concentrations were elevated in rats concurrently administered nicotine and methylated spirits compared with those given methylated spirits alone.

Table 2 Concentrations of nicotine (ng.mL⁻¹), ethanol (mg.dL⁻¹), methanol (mg.dL⁻¹), hormones (AVP, fmol.L⁻¹ & ALD, nmol.L⁻¹), plasma electrolytes (Na⁺ & K⁺, μmol), and pH in unanesthetized groups of rats administered every second consecutive day for 4 wk with nicotine and/or methylated spirits (n  =  8 in each group)

	Group
Parameter	Control	Nicotine	Methylated spirits	Nicotine + methylated spirits
Nicotine (ng.mL^−1)	nil	5.24 ± 0.35	nil	7.81 ± 0.21^†
Ethanol (mg.dL^−1)	nil	nil	68 ± 2	72 ± 1^‡
Methanol (mg.dL^−1)	nil	nil	35 ± 1	41 ± 1^‡
AVP (fmol.L^−1)	9.89 ± 0.35	8.56 ± 1.01	7.65 ± 0.25	6.72 ± 0.58^†
ALD (nmol.L^−1)	1.90 ± 0.15	3.80 ± 0.10	5.02 ± 0.19^†	6.18 ± 0.12^†
Plasma [Na^+] (mmol)	131 ± 1	140 ± 1	142 ± 1	145 ± 1^†
Plasma [K^+] (mmol)	4.32 ± 0.51	4.41 ± 0.24	4.27 ± 0.11	4.12 ± 0.13
Plasma pH	7.35 ± 0.03	7.30 ± 0.02	7.50 ± 0.01	7.40 ± 0.02

*p < 0.01 by comparison with control animals.

^†p < 0.01 by comparison with rats administered nicotine alone.

^‡p < 0.01 by comparison with rats administered methylated spirits alone.

Series 2: Renal Effects—Acute Indicator

Although urine voided dropped significantly below the control value, there was no translated alteration of plasma AVP levels (see Table 3). Urinary Na⁺ levels were significantly elevated in all treatment groups, and this was associated with attenuated ALD concentrations. Plasma Na⁺ values were significantly reduced in all treatment groups. Urinary K⁺ concentrations were markedly reduced in all treatment groups. Plasma K⁺ levels were significantly increased in animals infused with nicotine alone and in combination with methylated spirits. Plasma nicotine concentrations were increased in combined treatments. Plasma ethanol levels were significantly reduced and elevated in rats concurrently exposed to nicotine and methylated spirits, respectively.

Table 3 Total urine volume (mL), urine and plasma electrolytes (Na⁺ & K⁺, μmol), hormones (AVP, fmol.L⁻¹ & ALD, nmol.L⁻¹), concentrations of nicotine (ng.mL⁻¹), ethanol (mg.dL⁻¹), methanol (mg.dL^-1), and plasma pH in anesthetized groups of rats previously administered every second consecutive day for 4 wk. with nicotine and/or methylated spirits (n  =  8 in each group)

Group	Control	Nicotine	Methylated spirits	Nicotine + methylated spirits
Urine volume (mL)	36.74 ± 1.21	31.62 ± 1.02	30.53 ± 1.06	35.15 ± 1.54^†
Na^+ (μmol)	2351 ± 31	2740 ± 21	2935 ± 25*^†	2857 ± 19^†
K^+ (μmol)	890 ± 15	652 ± 19	688 ± 10*^†	660 ± 18^†
Plasma [Na^+] (mmol)	130 ± 1	126 ± 1	124 ± 1	125 ± 1
Plasma [K^+] (mmol)	4.02 ± 0.21	3.75 ± 0.16	4.35 ± 0.19^†	4.24 ± 0.11^†
AVP (fmol.L^−1)	9.42 ± 0.85	9.6 6± 1.05	9.75 ± 0.74	9.70 ± 1.12
ALD (nmol.L^−1)	1.90 ± 0.11	1.75 ± 1.11	1.68 ± 1.21*^†	1.71 ± 0.87^†
Nicotine (ng.mL^−1)	nil	4.84 ± 0.52	nil	6.88 ± 0.19^†
Ethanol (mg.dL^−1)	nil	nil	80 ± 1	68 ± 1^‡
Methanol (mg.dL^−1)	nil	nil	36 ± 1	45 ± 1^‡
Plasma pH	7.29 ± 0.04	7.33 ± 0.13	7.35 ± 0.08	7.40 ± 0.02

*p < 0.01 by comparison with control animals.

^†p < 0.01 by comparison with rats administered nicotine alone.

^‡p < 0.01 by comparison with rats administered methylated spirits alone.

Measurements of hematocrit were relatively stable in all groups approximating 41%. Mean arterial blood pressure (MAP) in all groups (range: 120 ± 1 to 123 ± 2 mmHg, p = 0.06) did not differ significantly from control animals (range: 122 ± 1 to 125 ± 1 mmHg, p  =  0.07). GFR in all treatment groups (nicotine: 3.29 ± 0.23; methylated spirits 3.33 ± 0.16; nicotine/methylated spirits 3.34 ± 0.52 mL.min⁻¹) did not differ from control animals (3.31 ± 0.41 mL.min⁻¹, p  =  0.08).

DISCUSSION

The principle objective of the current study was to explore the consequences of long-term ingestion of nicotine and methylated spirits on renal function in the Sprague-Dawley rat. The gavage method used in the current investigation was performed in order to avoid the unpalatability of administering nicotine via drinking water.[17]

The toxic effects of nicotine on renal function are well known, as emphasized in an early paper.[18] Methanol poisoning as a consequence of methylated spirits ingestion was published in communities resident in Papua New Guinea.[6] As far as the literature suggests, results from the current investigation are the first obtained investigating concurrent usage of nicotine and methylated spirits. It would be interesting to extend such observations to investigate the effects in human subjects.

The results in the present study suggest that concurrent administration of nicotine and methylated spirits over 4 wk. impairs renal fluid and electrolyte handling. The observations of nicotine on renal function in the current study supported findings[7] and discussions[11] in previous work.

Body weight gains were observed in animals administered methylated spirits alone or in combination with nicotine, whereas nicotine alone caused a loss of body weight (see Table 1). In animals infused with methylated spirits, it is possible that the energy from the ethanol contributed to weight gain[9] because progressive weight loss was observed in animals administered nicotine alone (see Table 1). Other studies demonstrated a depression in weight gain following short-term inhalation[19] and gavagal administration[20] of methanol. In the current study, the greater concentration of ethanol vs. methanol in the plasma of rats infused with methylated spirits alone or in combination with nicotine suggests a greater influence of the latter on hepatic metabolism.

Previous investigations have demonstrated that short-term cigarette smoke exposure leads to a reduction in body weight and food intake, suggesting a mechanism of alteration in activity of hypothalamic neuropeptide Y by nicotine.[21],[22] Others suggest that smoking-related health risks may increase during periods of significant weight loss.[23]

In unanesthetised animals, weekly Na⁺ excretion decreased in nicotine-infused animals but increased progressively in animals administered methylated spirits alone and in combination with nicotine (see Table 1). The elevated ALD levels in nicotine-administered rats were associated with the observed anti-natriuretic effect and subsequent Na⁺ retention (see Table 1 and 2). A possible explanation might be via increased nicotine-induced renal nerve activation translating into sodium retention by the kidney.[24] The loss of Na⁺ observed in conscious animals infused with methylated spirits alone and in combination with nicotine was in contrast to the renal Na⁺ retention observed in ethanol-administered [rats[10] attributable to an increase in Na⁺/K⁺ ATPase activity in the cortex and outer medulla.[25],[26] In the current study, in animals sacrificed at week 4, elevated aldosterone levels were associated with increased plasma concentrations of Na⁺ (see Table 2). This suggests a direct patho-physiological effect of methanol on renal function,[27] possibly at the level of the proximal and distal convoluted tubules to disrupt the ALD-mediated retention of Na⁺. Indeed, a study has shown the factors contributing to renal injury as hemolysis and myoglobinuria and hydropic changes in the proximal tubule.[28] It is interesting to note that alcohol dehydrogenase is responsible for the metabolism of methanol, and ethanol has a higher affinity for this enzyme and is therefore preferentially metabolized.[29] Competition thereof for metabolism would serve to elevate plasma concentrations of methanol, possibly contributing to the renal toxicity observed in the current investigation.

In conscious rats, the concentrations of nicotine, ethanol, and methanol were raised in the combined treated group (see Table 2). This suggests a possible competitive metabolism for a partially common detoxification process at sites on hepatic cytochrome P450. Indeed, the primary pathway of nicotine metabolism is P450 2A6-catalyzed 5′-oxidation.[30] It is important to realize that chronic nicotine administration induces hepatic ethanol-metabolizing CYP2E1, suggesting that nicotine use may increase the elimination of ethanol.[31] In the current study, however, this was not apparent, as plasma nicotine and ethanol were elevated in combined treatments (see Table 2), suggesting a competitive effect between methanol and nicotine for cytochrome metabolism. Indeed, methanol stimulates CYP2E1.[32] Plausible explanations for the reduction of plasma ethanol levels in the combined treatments (see Table 3) may be due to increased ethanol metabolism via CYP2E1, an isoform inducible by ethanol,[33],[34] and/or increased elimination of ethanol through nicotine-induced metabolic stimulation.[31]

Plasma nicotine concentrations rose by approximately 33% in week 4 after the sacrifice of rats concurrently administered nicotine and methylated spirits (see Table 2) and remained elevated by about 30% during vehicle infusion (see Table 3), by comparison with rats administered nicotine alone. Indeed, the possible inhibition of drug metabolism by competition for CYP2E1 may promote undesirable elevations in nicotine levels.

In vehicle-infused anesthetized animals, urinary Na⁺ excretion rate was increased in all groups (see Table 3). This finding was supported by investigations of nicotine on renal function in anesthetized dogs, whereby nicotine stimulates saluresis and diuresis, mechanisms mediated by the release of catecholamines from the adrenal medulla acting on beta-adrenergic receptors and increases in GFR.[35] The reduction of plasma ALD levels and increase in AVP in hypotonic saline-infused and nicotine-administered rats was possibly associated with gastric distension[36] and subsequent natriuretic and anti-diuretic responses (see Table 3).

The acute influence of nicotine infusion into the kidney at 1 mg.h⁻¹.kg⁻¹ bwt results in a fall in K⁺ reabsorption in the rat and rabbit[37] and no effect on K⁺ excretion in dogs infused nicotine at 0.5 μg.min⁻¹.kg⁻¹ bwt.[35] The results in the current study demonstrated no significant alteration in plasma K⁺ concentrations in conscious rats (see Table 2), although hypotonic saline loading decreased plasma K⁺ levels (see Table 3). Rather, we suggest an adaptive effect to nicotine loading in conscious animals. The attenuation of plasma K⁺ levels in anesthetized rats (see Table 3) cannot be explained in the current study, and is possibly attributable to experimental design.

In conclusion, the present study suggests that chronic exposure to methylated spirits alone and in combination with nicotine increases urinary Na⁺ loss. The renal toxicity is manifested hypothetically via elevations in plasma nicotine and methanol concentrations. This implies that patients who concurrently consume methylated spirits and smoke cigarettes have an increased risk of renal failure by being predisposed to fluid and electrolyte disturbances. The dangers implicit during methylated spirits consumption are obvious; nicotine can accelerate this progression resulting in failure of renal function.

ACKNOWLEDGMENT

Technical support in the laboratory and Animal House and a private loan were gratefully appreciated. Byk Gulden, Konstanz, is thanked for the donated trapanal.