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or ethanol administration
General Pharmacology 34 (2000) 43–51
Renal electrolyte and fluid handling in the rat following chloroquine and/or ethanol administration C.T. Musabayanea,*, R.G. Coopera, E. Osima, R.J. Balmentb a
Department of Physiology, University of Zimbabwe, P.O. Box MP 167, Mount Pleasant, Harare, Zimbabwe b School of Biological Sciences, Manchester University, Manchester M13 9PT, United Kingdom
Abstract We postulated that chloroquine and/or ethanol affect plasma arginine vasopressin (AVP) concentrations to alter renal function. Therefore, we studied the effects of chloroquine and/or ethanol on plasma AVP concentrations and fluid, urinary Na⫹ and K⫹ outputs in separate groups of anaesthetized Sprague-Dawley (SD) rats challenged with a continuous jugular infusion of 0.077 M NaCl at 150 l.min⫺1. After a 3-h equilibration period, vehicle, chloroquine (0.06 g.min⫺1), ethanol (2.4 or 24 g.min⫺1) or both chloroquine and ethanol were added to the infusate after 1 h (control) for 1 h 20 min (treatment). The animals were switched back to the infusate alone for the final 1 h 40 min recovery periods. Urine flow Na⫹ and K⫹ excretion rates were determined at 20-min intervals over the subsequent 4-h postequilibration period. Blood was collected from separate groups of animals at the end of treatment period or equivalent time for control animals for measurement of plasma aldosterone and AVP concentrations by radioimmunoassay. Simultaneous chloroquine and ethanol infusion significantly (p ⬍ 0.01) increased plasma chloroquine concentrations in an ethanol dose-dependent manner by comparison with animals administered chloroquine alone. Chloroquine infusion alone (0.06 g.min⫺1) and/or ethanol (2.4 or 24 g.min⫺1) elevated plasma AVP concentrations from 9.73 ⫾ 1.64 fmol.l⫺1 in control rats to 15.65 ⫾ 2.49 fmol.l⫺1, 17.39 ⫾ 4.21 fmol.l⫺1, and 33.87 ⫾ 6.18 fmol.l⫺1, respectively. Separate administration of chloroquine or ethanol at low dose rates increased urinary Na⫹ excretion rates. We conclude that the impairment of renal electrolyte handling associated with chloroquine administration may be exacerbated by ethanol. 2000 Elsevier Science Inc. All rights reserved. Keywords: Ethanol; Chloroquine; Kidney; Sodium
1. Introduction Current evidence suggests that chloroquine used in the treatment and management of malaria and for the treatment of rheumatoid arthritis (Augustijns et al., 1992; Augustijns and Verbeke, 1993) can impair kidney function (Musabayane et al., 1993; Musabayane et al., 1994). In the rat, acute chloroquine administration increases plasma arginine vasopressin (AVP) concentration, which appears to be responsible for the associated increases in renal Na⫹ excretion (Musabayane et al., 1996). Increases in the plasma levels of AVP after acute ethanol administration have been reported (Colantonio et al., 1991), resulting in renal fluid retention (Taivainen
* Corresponding author. Tel.: 263-4-333678; fax: 263-4-333678/ 333407. E-mail address: [email protected] or ctm@kidney. uz.zw (C.T. Musabayane).
et al., 1995). However, it has also been shown that ethanol not only reduces plasma AVP concentration, but also alters AVP-induced renal water permeability (Eisenhofer and Johnson, 1982; Ray et al., 1992; Carney et al., 1995). Thus, the effects of alcohol on renal electrolyte and fluid excretion remain unclear. Therefore, the current study was designed to investigate the effects of acute chloroquine and/or ethanol administration on renally active hormones in rats. In addition, we envisaged establishing not only the effects of ethanol on renal electrolyte excretion, but also the influence of alcohol on the renal effects of chloroquine. In malaria endemic areas, it is not uncommon to find some individuals on chloroquine treatment or prophylaxis for malaria consuming alcohol regularly (personal observation or confession). In vitro studies in renal brush border membranes suggest that ethanol impairs Na⫹ reabsorption (Parenti et al., 1991; Rodrigo et al., 1998), which may explain electrolyte deficiency syndromes described in chronic alcoholics (Heaton et al., 1962; De Marchi et
0306-3623/00/$ – see front matter 2000 Elsevier Science Inc. All rights reserved. PII: S0306-3623(00)00045-8
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al., 1993). So it is conceivable that alcohol consumption and chloroquine administration could interact to impair renal function in these patients. The hypotonic salineinfused rat protocol used in the present study is a standard preparation used in many studies to assess the adequacy of renal function in response to mild fluid and salt challenge (Musabayane et al., 1996). 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (300–350 g) bred and housed in the Medical Faculty Animal House at the University of Zimbabwe were used in the study. The rats were maintained on a 12-h light/12-h dark regime. Ethical clearance for these studies was provided by the Ministry of Agriculture, Zimbabwe, as stipulated in the Scientific Animal Experiment Act, 1963. 2.2. Renal function studies The animals were anaesthetized by intraperitoneal injection of Trapanal (sodium 5-ethyl-5⬘-(1-methylbutyl)-2-thio-barbiturate; Byk Gulden, Konstanz, Germany) at 0.11 g.kg⫺1 body weight and challenged with hypotonic saline (0.077 M NaCl) infusion. 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, U.S.A.) to allow intravenous infusion of 0.077 M NaCl. The left carotid artery was also cannulated with polythene tubing (i.d., 0.58 mm and o.d., 0.96mm; Clay Adams) and then connected to a blood pressure transducer (Grass Polygraph, Model 790; Grass Instruments Company, Quincy, MA) 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 at 37 ⫾ 1⬚C with a heated table. Rats were placed on a continuous infusion of 0.077 M NaCl at 150 l.min⫺1 (Sage Syringe Pump Model 351), and a 3-h equilibration period was allowed. After this, consecutive 20-min urine collections were made into pre-weighed plastic vials over the subsequent 4 h of 1 h control, 1 h 20 min treatment, and 1 h 40 min posttreatment periods for measurements of urine osmolalities and flow rates and Na⫹ and K⫹ excretion rates. The effects of chloroquine or ethanol were examined in separate groups of rats in which chloroquine (0.06 g.min⫺1) and/or ethanol at either 2.4 or 24 g.min⫺1 were added to the infusate during the 1 h 20 min treatment period, resulting in total dose of chloroquine of 16 g.kg⫺1 and ethanol 0.64 or 6.4 mg.kg⫺1. The animals were switched back to the infusate alone for the final 1 h 40 min recovery periods. A terminal blood sample (2 ml) was collected by cardiac puncture for determination of osmolality.
2.3. Glomerular filtration rate measurements Glomerular filtration rate (GFR) was determined from the clearance of inulin in the same animals prepared for renal studies. All animals were given a priming dose (0.3 Ci in 0.3 ml saline) of 3[H] inulin (Amersham, Buckinghamshire, UK; specific activity 1.74 Ci.mmol⫺1) and then placed on a continuous intravenous infusion at 150 l.min⫺1 of 0.077 M NaCl containing inulin (0.14 Ci.min⫺1) throughout the experimental period. Blood samples (200 l) were taken at 1-h intervals into heparinized haematocrit tubes throughout the 4-h postequilibration period for measurement of haematocrit before analysis of separated plasma. Aliquots of urine (10 l) and separated plasma (10 l) were counted on a Minaxi  Tri-Carb 4000CA series liquid Scintillation Counter (Packard Instrument Co., Downer’s Grove, IL). 2.4. Analytical methods 2.4.1. Measurement of electrolytes and osmolality Urine volume was determined gravimetrically. Na⫹ and K⫹ were determined by Flame Photometry (Corning model 435 Flame Photometer; Corning Limited, Halstead, UK). The fractional excretion rates of sodium ⫹ ) were calculated. Osmolalities of urine and plasma (FENa were measured on 100-l samples by freezing point depression using a Camlab osmometer (Cambridge, UK). 2.4.2. Plasma ethanol, chloroquine, and hormone measurements Blood was collected from parallel groups of animals prepared for renal studies by cardiac puncture in cooled heparinized containers at the end of 1 h 20 min treatment or corresponding time for control animals. Blood was placed and spun in a Sorvall RT6000 Refrigerated Centrifuge (Dupont, Newtown, MA) at 2,500 g.min⫺1 for 10 min at ⫺4⬚C, and separated plasma was stored at ⫺20⬚C until measurement of aldosterone and AVP. Measurements for ethanol and chloroquine were performed by 12 h after separation. Plasma ethanol was measured by gas chromatography. Chloroquine concentration was assayed after extraction. Initially, 0.5 ml of potassium hydroxide (16 M) was added to a separator flask containing 4 ml plasma, followed by 25 ml chloroform. The mixture was well shaken. Subsequently, chloroquine was extracted from the lower chloroform layer (21 ml) with 4 ml hydrochloric acid (1 M) and measured by UV Spectroscopy (Shimadzu U-V-Visible160A) at 343 nm spectrophotometrically using plasma as a blank. Plasma aldosterone concentration was measured by Coat-A-Count procedure using a kit from Diagnostic Products Corporation (Los Angeles, CA). This is a solid-phase radioimmunoassay procedure based on aldosterone-specific antibody immobilized to the wall of a polypropylene tube. The lower limit of detection was
C.T. Musabayane et al. / General Pharmacology 34 (2000) 43–51
44 fmol.l⫺1. Inter- and intra-assay coefficients of variation were 8.1% (n ⫽ 20) and 8.3% (n ⫽ 20), respectively. AVP was determined as described by Forsling and Peysner (1988) using the DSL-1800 Arginine Vasopressin Radioimmunoassay kit from Diagnostic Systems Laboratories (Webster, TX). Vasopressin was extracted from plasma using Sep Pak C18 cartridges (Millipore Water Associates, Harrow, UK). The lower limit of detection was 0.5 fmol.l⫺1 and intra- and interassay variations were 7.7% (n ⫽ 12) and 11.9% (n ⫽ 12), respectively. 2.4.3. Data presentation and statistical analysis Values are presented as means ⫾ SEM. Data in control rats were used as a baseline for comparison. Renal electrolyte excretion and urine flow rate are presented graphically showing 20-min collections over the 4-h postequilibration period. The total fluid voided, urine osmolalities, and Na⫹ and K⫹ excreted during the 1 h 20 min of chloroquine and/or ethanol administration were compared to that of control animals at the corresponding time. The plasma concentrations of ethanol, chloroquine, and hormones data from treated animals were compared to control values to evaluate the effects of each treatment. All data were subjected to one-way
45
analysis of variance and Scheffe’s multiple comparison was used to resolve any apparent differences. A value of p ⬍ 0.05 was considered significant. 3. Results 3.1. Urinary effects of chloroquine and/or ethanol The urine flow and Na⫹ excretion rates in vehicleinfused control animals during the 4-h postequilibration period stabilized at rates that closely approximated the rates of infusion (Fig. 1, A and B). The urine flow rate ranged from 141 to 163 l.min⫺1 compared with the infusion rate of 150 l.min⫺1. Na⫹ excretion rates stayed around 9 mol.min⫺1, a value that was not significantly different from the infusion rate (11.6 mol.min⫺1). K⫹ excretion rates were stable, approximating 3.5 mol.min⫺1 throughout the postequilibration period. Administration of chloroquine for 1 h 20 min did not affect urine flow rate (Fig. 1C). However, it did affect Na⫹ excretion rate, which increased to 13.1 ⫾ 0.9 mol.min⫺1 (n ⫽ 8) 20 min after the commencement of chloroquine administration, a value that was significantly (p ⬍ 0.01) higher than that of controls (9.1 ⫾ 0.4 mol.min⫺1, n ⫽ 8) for the corresponding period (cf.
Fig. 1. Urine flow and Na⫹ and K⫹ excretion rates in 0.077 M NaCl infused control rats (A and B) and rats administered chloroquine (Chq) at 0.06 g.min⫺1 (C and D) for 1 h 20 min. Values are presented as means for each 20-min collection for the 4-h postequilibration period; vertical bars indicate SE of means.
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Fig. 2. The urine flow, Na⫹ and K⫹ excretion rates in rats administered ethanol (EtoH) at dose rates at either 2.4 g.min⫺1 (A and B) or 24 g.min⫺1 (C and D). Values are presented for each 20-min collection for the 4-h postequilibration period; vertical bars indicate SE of means.
Fig. 1, B and D). K⫹ excretion rates were not altered by this treatment. In comparison to control animals, ethanol administration at both dose rates (2.4 or 24 g.min⫺1) reduced urine flow rate, resulting in less volume of urine voided during treatment period (See Fig. 2 and Table 1). The reduction in urine flow rate was maintained during the recovery period in animals treated with the higher dose (cf. Fig. 2, A and C). Ethanol infused at the low dose rate increased urinary Na⫹ excretion rates and the total amount excreted, but the total amount of K⫹ excreted was reduced (Table 1). Ethanol at 24 g.min⫺1 increased Na⫹ excretion rate to 15.51 ⫾ 0.68 mol.min⫺1 by 20
min, but the rate subsequently dropped throughout the treatment period to reach 6.94 ⫾ 0.86 mol.min⫺1 by the last treatment. The total amount of Na⫹ excreted during the treatment compared to that excreted by control animals at the corresponding time (Table 1), while urinary K⫹ output was reduced. The administration of combined chloroquine and ethanol at a lower dose (2.4 g.min⫺1) decreased diuresis, which reached 87 l.min⫺1 20 min after the cessation of ethanol infusion (Fig. 3A). The total volume of fluid voided during the treatment period was significantly less than that of control animals (see Table 1). However, combined administration of chloroquine and etha-
Table 1 Comparison of terminal plasma osmolalities, total amounts of urine voided, urine osmolalities, Na⫹ and K⫹ excreted during the 1 h 20 min treatment period and in groups of animals administered chloroquine and/or ethanol with control rats Treatment
UV (ml)
Control Chq (0.06 g.min⫺1) EtoH (2.4 g.min⫺1) EtoH (24 g.min⫺1) Chq ⫹ EtoH (2.4 g.min⫺1) Chq ⫹ EtoH (24 g.min⫺1)
11.82 11.59 8.55 6.08 7.46 11.20
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.03 1.16 0.33* 0.19* 0.14* 0.49
Na⫹ (mol) 842 1054 1140 817 883 839
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
K⫹ (mol) 51 44* 22* 39 49 19
* p ⬍ 0.01 by comparison with control animals. n ⫽ 8 in all groups.
275 265 153 176 149 191
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
20 22 10* 17* 26* 35*
Urine osmolality (mOsm.kg⫺1 H2O) 285 274 161 152 160 265
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
21 25 14* 17* 10* 23
Plasma osmolality (mOsm.kg⫺1 H2O) 279 284 263 228 255 273
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
9 5 3* 3* 6* 9
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Fig. 3. The urine flow, Na⫹ and K⫹ excretion rates in rats simultaneously administered chloroquine (Chq) at 0.06 g.min⫺1 and ethanol (EtoH) at either 2.4 g.min⫺1 (A and B) or 24 g.min⫺1 (C and D). Values are presented for each 20-min collection for the 4-h postequilibration period; vertical bars indicate SE of means.
nol at a higher dose (24 g.min⫺1) was associated with temporary diuresis, which reached 114 l.min⫺1 by 40 min after the start of infusion before rising to values that did not statistically differ from control animals at corresponding times (Fig. 3C). The total volume of fluid voided during the treatment period did not significantly differ from values observed in control animals at the corresponding time. Na⫹ excretion rates were not significantly altered by the administration of chloroquine and ethanol (at all doses) when compared to control, while K⫹ excretion was reduced (Fig. 3, B and D). 3.2. Urine osmolality and FENa⫹ rate during the treatment period and terminal plasma osmolality The mean FENa⫹ rate during chloroquine infusion was not significantly altered when compared to values of control rats at the corresponding time (Fig. 4C). However, ethanol alone at a low dose significantly (p ⬍ 0.05) increased the mean FENa⫹ rate (Fig. 4C). Combined chloroquine and ethanol at all doses did not significantly change the mean FENa⫹ rate. The urine osmolalities during the administration of chloroquine alone or in combination with ethanol at a high dose were not significantly different from that of control animals at the corresponding time (Table 1). However,
urine osmolalities in animals administered ethanol alone at both doses or ethanol at a low dose in combination with chloroquine were significantly (p ⬍ 0.01) low by comparison with control rats. The mean differences in FENa⫹ rates and urine osmolalities in the 1 h 20 min of chloroquine and/or ethanol administration were reflected in plasma osmolalities for samples collected at the end of the experiment with animals administered ethanol alone or chloroquine with ethanol at a low dose exhibiting low values (Table 1). 3.3. Plasma hormones, ethanol and chloroquine concentrations, mean arterial blood pressure, and GFR Measurements of haematocrit were relatively stable in all groups of animals, remaining at approximately 42% throughout the 4-h postequilibration period. Fig. 4 shows that the mean arterial blood pressure was significantly (p ⬍ 0.01) elevated above the pretreatment values by acute ethanol infusion at either 2.4 g.min⫺1 (from 129 ⫾ 2 mmHg to 137 ⫾ 1 mmHg) or 24 g.min⫺1 (from 126 ⫾ 1 mmHg to 132 ⫾ 1 mmHg). The increases were associated with an increase in GFR to approximately 4.0 ml.min⫺1, a value significantly higher than that observed during the pretreatment (approximately 3.0 ml.min⫺1). Infusion of chloroquine alone or in com-
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Fig. 4. Mean arterial blood pressure (A), GFR (B), and FeNa⫹ (C) rates in control rats and rats administered chloroquine and/or ethanol (EtoH). Values are calculated to indicate 1-h control (pretreatment), 1 h 20 min of treatment, and 1 h 40 min of recovery for the 4-h postequilibration period; vertical bars indicate SE of means.
bination with ethanol did not have any significant effects on either mean arterial blood pressure or GFR. Table 2 shows that chloroquine infusion for 1 h 20 min significantly (p ⬍ 0.01) increased plasma AVP concentrations by comparison with control animals. The AVP levels were further elevated when chloroquine was co-administered with ethanol in an ethanol dosedependent manner. By comparison with animals administered chloroquine alone, plasma chloroquine concentrations were increased between 97 and 167% following 1 h 20 min of combined chloroquine and ethanol at both doses. Plasma ethanol concentrations were not detectable after 1 h 20 min administration of chloroquine and/ or ethanol at the low dose administration rate (2.4 g.min⫺1). Simultaneous chloroquine and ethanol infusion significantly (p ⬍ 0.01) increased plasma chloroquine concentrations in an ethanol dose-depen-
dent manner by comparison with animals administered chloroquine alone. Plasma ethanol concentrations were detectable in groups of rats administered ethanol at 24 g.min⫺1 alone or in combination with chloroquine. The mean plasma ethanol concentrations following simultaneous infusion of chloroquine and ethanol at a high dose were elevated by 26% by comparison with that of ethanol alone. In contrast, plasma ethanol concentrations were not detectable in animals following infusion of ethanol alone at a low dose or in combination with chloroquine. 4. Discussion The results of the current study extend our previous observations that acute chloroquine administration increases plasma AVP concentrations (Musabayane et
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Table 2 Plasma chloroquine, ethanol, aldosterone, and AVP concentrations in control rats and in animals administered chloroquine and/or ethanol for 1 h 20 min
Control Chq (0.6 g.min⫺1) EtoH (2.4 g min⫺1) EtoH (24 g min⫺1) Chq ⫹ EtoH (2.4 g min⫺1) Chq ⫹ EtoH (24 g min⫺1)
Ethanol (mg.dl⫺1)
Chloroquine (g. ml⫺1)
Aldosterone (nmol.l⫺1)
not detected not detected not detected 39.32 ⫾ 3.90 not detected 49.60 ⫾ 3.35
not measured 2.89 ⫾ 0.22 not measured not measured 5.70 ⫾ 0.43* 7.71 ⫾ 0.83*,**
1.86 1.77 1.83 1.79 1.77 2.23
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.20 0.19 0.09 0.29 0.29 0.24*
AVP (fmol.l⫺1) 9.73 15.65 18.46 29.70 17.29 33.87
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
1.64 2.49* 3.42* 4.32* 4.21* 6.18*
* p ⬍ 0.01 by comparison with control animals. ** p ⬍ 0.01 by comparison with the group administered Chq ⫹ EtoH (2.4 g min⫺1). n ⫽ 8 in all groups.
al., 1996) by showing that this effect is further enhanced when the antimalarial is co-administered with ethanol. The dose rate for intravenous chloroquine infusion resulting in total dose of 16 g.kg⫺1 was significantly (p ⬍ 0.01) lower than doses previously used in rats (600–1500 g.kg⫺1; Adelusi et al., 1982) and humans (750–850 g.kg⫺1; White et al., 1988). The administration dose rates of ethanol, which resulted in total doses of 0.64 or 6.4 mg.kg⫺1, were much lower than doses previously used in rats (1.2–2.0 g.kg⫺1 Wu et al., 1992). Ethanol administration caused dose-dependent increases in plasma AVP concentrations and responses in renal Na⫹ handling. The low dose increased the total amounts of urinary Na⫹ loss, but the high dose did not significantly affect the total urinary Na⫹ loss despite eliciting high excretion rates in the initial 20 min (see Fig. 2D). We suggest adaptive changes in response to the initial elevated urinary Na⫹ loss led to subsequent Na⫹ retention by 40 min of treatment. This possibly contributed to the differences in the total amounts of urinary Na⫹ after infusion of ethanol at either low or high dose rate. Dose-dependent effects of ethanol on (Na⫹-K⫹)-ATPase activity, an enzyme that increases Na⫹ transport in renal collecting ducts, may also be invoked to explain the differences. For instance Rodrigo et al. (1998) have reported ethanol dose-dependent effects on the (Na⫹-K⫹)-ATPase. Indeed, we have reported Na⫹ retention after administration of ethanol at 60 g.kg⫺1 (Musabayane et al., 1985). Chloroquine and/ or ethanol administration led to significant reduction in urinary K⫹ loss. The hormonal basis, if any, of this retention remains unresolved. Previously, we attributed urinary Na⫹ loss after acute chloroquine infusion to elevated plasma AVP levels (Musabayane et al., 1993). However, combined administration of chloroquine and ethanol at both doses did not affect renal Na⫹ excretion despite inducing significant increases in plasma AVP concentrations (Table 2). This suggests the ethanol inhibition of the chloroquine-induced renal Na⫹ loss cannot be ascribed to ethanol influencing plasma AVP concentrations. Presumably, ethanol induced alteration in membrane fluidity (Villanueva et al., 1994), and direct interference with
functions of transport proteins in renal brush border (Deves and Kripka, 1990) altered the renal effects of chloroquine. We cannot exclude the influence of chloroquine or ethanol on the autonomic nervous system by a direct action on tubular reabsorption mechanisms or an indirect action on renal haemodynamics to affect renal electrolyte excretion. For instance the reported stimulation of the sympathetic nervous system by ethanol (Delarue et al., 1997) can increase blood pressure and, if this is coupled with a rise in GFR, can increase renal electrolyte excretion. Indeed, ethanol alone increased mean arterial blood pressure by 30%, and this was associated with an increase in GFR by comparison with control animals. However, chloroquine has an antimuscarinic effect (Lot and Bennet, 1982; Mubagwa and Adler, 1988) and as such would be expected to reduce sympathetic responses to cause renal Na⫹ retention. Therefore, renal electrolyte excretion after chloroquine and/or ethanol administration can partly be attributed to the influence of chloroquine or ethanol on the sympathetic nervous system. Acute ethanol at both doses resulted in antidiuresis, perhaps because of high plasma AVP levels. However, some studies suggest that ethanol ingestion acutely alters water balance with increased diuresis caused by inhibition of AVP release (Eisenhofer and Johnson, 1982). The absence of antidiuresis despite increases in plasma AVP concentrations after the administration of chloroquine alone or with ethanol is perhaps caused by inhibition of cAMP production by chloroquine. We have observed in isolated rat inner medullary collecting ducts that chloroquine inhibits the AVP-induced cAMP production that mediates the antidiuretic effects of AVP (Musabayane et al., 1997). The plasma ethanol concentrations after infusion of a lower dose of ethanol alone or in combination with chloroquine were undetectable, but were observed after infusion of ethanol at high doses alone or in combination with chloroquine. Perhaps the ability of hepatic catalase to rapidly metabolize low ethanol concentration (Lieber, 1997) accounted for these differences. Plasma chloroquine and ethanol concentrations were
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significantly elevated in animals when chloroquine and ethanol were simultaneously administered, by comparison with groups of animals administered either chloroquine alone or ethanol alone. This suggests reduction in chloroquine or ethanol metabolism following coadministration of these compounds. Presumably chloroquine and ethanol compete for a partially common detoxification process, which includes binding with cytochrome P450. The cytochrome P450 system involves not only the ethanol oxidizing system (Lieber, 1997), but also the detoxification process of chloroquine (Thabrew and Ioannides, 1984). This would be of clinical importance if this was established because simultaneous intake of chloroquine and ethanol in areas where malaria is endemic may exacerbate the reported impairment of renal electrolyte handling associated with chloroquine administration in the rat (Musabayane et al., 1996) and man (Musabayane et al., 1999). Since we have shown no significant differences in renal function between anesthetized and conscious salineinfused preparations (Balment et al., 1984; Brimble et al., 1986), it is possible that the effects of chloroquine and/or ethanol on renal electrolyte and fluid handling seen in the present study may apply to awake conditions. In summary, results of the present study confirm that the pathophysiology of renal regulation of water and electrolytes after chloroquine and/or ethanol administration may be commonly ascribed to altered plasma AVP concentrations, but may also be caused by other intrarenal effects such as alterations in GFR. The results also suggest dose dependent responses on renal electrolyte excretion in response to ethanol.
Acknowledgments This study was funded by the University of Zimbabwe Research Board (YYH010/3580) and The Kapnek Charitable Trust. The authors thank Byk Gulden, Konstanz, for the gift of Trapanal.
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action of arginine vasopressin in the conscious unrestrained rat. Acta Endocrinol (Copen) 119, 386–390. Carney, S.L., Gillies, A.H.B., Ray, C.D., 1995. Acute effect of ethanol on renal electrolyte transport in the rat. Clin Exp Pharmacol Physiol 22, 629–634. Colantonio, D., Casale, R., Desiati, P., De Michele, G., Mammarella, M., Pasqualetti, P., 1991. A possible role of atrial natriuretic peptide in ethanol-Induced acute diuresis. Life Sci 48, 635–642. Delarue, J., Schneiter, P., Henry, S., Cayeux, C., Haesler, E., Jequier, E., Tappy, L., 1997. Effects of adrenergic blockade on hepatic glucose production during ethanol administration. Clin Physiol 17 (5), 509–521. De Marchi, S., Cerchin, E., Basile, A., Bertotti, A., Nardini, R., Bartoli, E., 1993. Dose- and time-dependant cardiovascular responses induced by ethanol. J Pharmacol Exp Ther 268 (1), 78–84. Deves, R., Kripka, R.M., 1990. Inhibition of choline transport in erythrocytes by n-alkanols. Biochim Biophys Acta 1030, 32–40. Eisenhofer, G., Johnson, R.H., 1982. Effect of ethanol ingestion on plasma vasopressin and water balance in humans. Am J Physiol 242, R522–R527. Forsling, M.L., Peysner, K., 1988. Pituitary and plasma vasopressin concentration and fluid balance throughout the oestrous period. J Endocrinol 117, 397–402. Heaton, F.W., Rynah, L.N., Beresford, C.C., Bryson, R.W., Martin, T.F., 1962. Hypomagnesaemia in chronic alcoholism. Lancet 2, 802–805. Lieber, C.S., 1997. Pharmacology and therapeutics: alcohol liver disease. In: Friedman, G., Jacobson, E.D., McCallum, R.W. (Eds.), Gastrointestinal pharmacology and therapeutics. LippincottRaven Press, Philadelphia, PA, pp. 465–487. Lot, T.Y., Bennett, T., 1982. Comparison of the effects of chloroquine quinacrine and quinidine on autonomic neuroeffector mechanisms. Med Biol 60 (6), 307–315. Mubagwa, K., Adler, J., 1988. Muscarinic antagonist action of clinical doses of chloroquine in healthy volunteers. J Autonomic Nervous System 24, 147–155. Musabayane, C.T., Brimble, M.J., Balment, R.J., 1985. Renal sodium retention and vasopressin induced kaliuresis in ethanol anaesthetised rats. Acta Endocrinol (Copen) 110, 214–220. Musabayane, C.T., Ndhlovu, C.E., Mamutse, G., Bwititi, P., Balment, R.J., 1993. Acute chloroquine administration increases renal sodium excretion. J Trop Med Hyg 96, 305–310. Musabayane, C.T., Ndhlovu, C.E., Balment, R.J., 1994. The effects of oral chloroquine administration on kidney function. Renal Failure 16 (2), 221–228. Musabayane, C.T., Windle, R.J., Forsling, M.L., Balment, R.J., 1996. Arginine vasopressin mediates the chloroquine induced increase in renal sodium excretion. Trop Med Int Health 1 (4), 542–550. Musabayane, C.T., Wargent, E.T., Balment, R.J., 1997. Chloroquine inhibits the antidiuretic effects of AVP by reducing cAMP formation in the inner medullary collecting ducts. J Endocrinol 152 (Suppl.), 45. Musabayane, C.T., Musvibe, A., Wenyika, J, Munjeri, O., Osim, E.E., 1999. Chloroquine influences renal function in rural Zimbabweans with acute transient fever. Renal Failure 21 (2), 189–197. Parenti, P., Giordana, B., Hanozet, G.M., 1991. In vitro effect of ethanol on sodium and glucose transport in rabbit renal brush border membrane vesicles. Biochim Biophys Acta 1070, 92–98. Ray, C., Carney, S.L., Gillies, A.H.B., 1992. Effect of ethanol on water and chloride transport in the rat papillary collecting duct. Miner Electrolyte Metab 18, 370–374. Rodrigo, R., Thielemann, L., Orellana, M., 1998. Acute and chronic effect of ethanol on (Na⫹ K)-ATPase activity and cyclic AMP response to vasopressin in rat papillary collecting duct cells. Gen Pharmacol 30 (5), 663–667.
C.T. Musabayane et al. / General Pharmacology 34 (2000) 43–51 Taivainen, H., Laitinen, K., Ta¨htela¨, R., Kiianmaa, K., Va¨hima¨ki, M.J., 1995. Role of plasma vasopressin in changes of water balance accompanying acute alcohol intoxication. Alcohol Clin Exp Res 19 (3), 759–762. Thabrew, M.I., Ioannides, C., 1984. Inhibition of rat hepatic mixed function oxidases by antimalarial drugs: selectivity for cytochrome P450 and P448. Chem Biol Int 51, 285–294. Villanueva, J., Chandler, C.J., Shimasaki, N., Tang, A.B., Nakamura, M., Phinney, S.D., Halstead, C.H., 1994. Effects of ethanol feeding
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on liver, kidney and jejunal membranes of micropigs. Hepatology 19 (5), 1229–1240. White, N.J., Miller, K.D., Churchill, F.C., Churchill, F.C., Berry, C., Brown, J., Williams, S.B., Greenwood, B.M., 1988. Chloroquine treatment of severe malaria in children: pharmacokinetics, toxicity and dosage recommendations. New Eng J Med 319, 1493–1500. Wu, P.H., Liu, J.-F., Mihic, S.J., Kalant, H., 1992. Arginine-8-vasopressin potentiates acute ethanol intoxication. Pharmacol Biochem Behav 41, 409–414.
Renal electrolyte and fluid handling in the rat following chloroquine and/or ethanol administration C.T. Musabayanea,*, R.G. Coopera, E. Osima, R.J. Balmentb a
Department of Physiology, University of Zimbabwe, P.O. Box MP 167, Mount Pleasant, Harare, Zimbabwe b School of Biological Sciences, Manchester University, Manchester M13 9PT, United Kingdom
Abstract We postulated that chloroquine and/or ethanol affect plasma arginine vasopressin (AVP) concentrations to alter renal function. Therefore, we studied the effects of chloroquine and/or ethanol on plasma AVP concentrations and fluid, urinary Na⫹ and K⫹ outputs in separate groups of anaesthetized Sprague-Dawley (SD) rats challenged with a continuous jugular infusion of 0.077 M NaCl at 150 l.min⫺1. After a 3-h equilibration period, vehicle, chloroquine (0.06 g.min⫺1), ethanol (2.4 or 24 g.min⫺1) or both chloroquine and ethanol were added to the infusate after 1 h (control) for 1 h 20 min (treatment). The animals were switched back to the infusate alone for the final 1 h 40 min recovery periods. Urine flow Na⫹ and K⫹ excretion rates were determined at 20-min intervals over the subsequent 4-h postequilibration period. Blood was collected from separate groups of animals at the end of treatment period or equivalent time for control animals for measurement of plasma aldosterone and AVP concentrations by radioimmunoassay. Simultaneous chloroquine and ethanol infusion significantly (p ⬍ 0.01) increased plasma chloroquine concentrations in an ethanol dose-dependent manner by comparison with animals administered chloroquine alone. Chloroquine infusion alone (0.06 g.min⫺1) and/or ethanol (2.4 or 24 g.min⫺1) elevated plasma AVP concentrations from 9.73 ⫾ 1.64 fmol.l⫺1 in control rats to 15.65 ⫾ 2.49 fmol.l⫺1, 17.39 ⫾ 4.21 fmol.l⫺1, and 33.87 ⫾ 6.18 fmol.l⫺1, respectively. Separate administration of chloroquine or ethanol at low dose rates increased urinary Na⫹ excretion rates. We conclude that the impairment of renal electrolyte handling associated with chloroquine administration may be exacerbated by ethanol. 2000 Elsevier Science Inc. All rights reserved. Keywords: Ethanol; Chloroquine; Kidney; Sodium
1. Introduction Current evidence suggests that chloroquine used in the treatment and management of malaria and for the treatment of rheumatoid arthritis (Augustijns et al., 1992; Augustijns and Verbeke, 1993) can impair kidney function (Musabayane et al., 1993; Musabayane et al., 1994). In the rat, acute chloroquine administration increases plasma arginine vasopressin (AVP) concentration, which appears to be responsible for the associated increases in renal Na⫹ excretion (Musabayane et al., 1996). Increases in the plasma levels of AVP after acute ethanol administration have been reported (Colantonio et al., 1991), resulting in renal fluid retention (Taivainen
* Corresponding author. Tel.: 263-4-333678; fax: 263-4-333678/ 333407. E-mail address: [email protected] or ctm@kidney. uz.zw (C.T. Musabayane).
et al., 1995). However, it has also been shown that ethanol not only reduces plasma AVP concentration, but also alters AVP-induced renal water permeability (Eisenhofer and Johnson, 1982; Ray et al., 1992; Carney et al., 1995). Thus, the effects of alcohol on renal electrolyte and fluid excretion remain unclear. Therefore, the current study was designed to investigate the effects of acute chloroquine and/or ethanol administration on renally active hormones in rats. In addition, we envisaged establishing not only the effects of ethanol on renal electrolyte excretion, but also the influence of alcohol on the renal effects of chloroquine. In malaria endemic areas, it is not uncommon to find some individuals on chloroquine treatment or prophylaxis for malaria consuming alcohol regularly (personal observation or confession). In vitro studies in renal brush border membranes suggest that ethanol impairs Na⫹ reabsorption (Parenti et al., 1991; Rodrigo et al., 1998), which may explain electrolyte deficiency syndromes described in chronic alcoholics (Heaton et al., 1962; De Marchi et
0306-3623/00/$ – see front matter 2000 Elsevier Science Inc. All rights reserved. PII: S0306-3623(00)00045-8
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al., 1993). So it is conceivable that alcohol consumption and chloroquine administration could interact to impair renal function in these patients. The hypotonic salineinfused rat protocol used in the present study is a standard preparation used in many studies to assess the adequacy of renal function in response to mild fluid and salt challenge (Musabayane et al., 1996). 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (300–350 g) bred and housed in the Medical Faculty Animal House at the University of Zimbabwe were used in the study. The rats were maintained on a 12-h light/12-h dark regime. Ethical clearance for these studies was provided by the Ministry of Agriculture, Zimbabwe, as stipulated in the Scientific Animal Experiment Act, 1963. 2.2. Renal function studies The animals were anaesthetized by intraperitoneal injection of Trapanal (sodium 5-ethyl-5⬘-(1-methylbutyl)-2-thio-barbiturate; Byk Gulden, Konstanz, Germany) at 0.11 g.kg⫺1 body weight and challenged with hypotonic saline (0.077 M NaCl) infusion. 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, U.S.A.) to allow intravenous infusion of 0.077 M NaCl. The left carotid artery was also cannulated with polythene tubing (i.d., 0.58 mm and o.d., 0.96mm; Clay Adams) and then connected to a blood pressure transducer (Grass Polygraph, Model 790; Grass Instruments Company, Quincy, MA) 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 at 37 ⫾ 1⬚C with a heated table. Rats were placed on a continuous infusion of 0.077 M NaCl at 150 l.min⫺1 (Sage Syringe Pump Model 351), and a 3-h equilibration period was allowed. After this, consecutive 20-min urine collections were made into pre-weighed plastic vials over the subsequent 4 h of 1 h control, 1 h 20 min treatment, and 1 h 40 min posttreatment periods for measurements of urine osmolalities and flow rates and Na⫹ and K⫹ excretion rates. The effects of chloroquine or ethanol were examined in separate groups of rats in which chloroquine (0.06 g.min⫺1) and/or ethanol at either 2.4 or 24 g.min⫺1 were added to the infusate during the 1 h 20 min treatment period, resulting in total dose of chloroquine of 16 g.kg⫺1 and ethanol 0.64 or 6.4 mg.kg⫺1. The animals were switched back to the infusate alone for the final 1 h 40 min recovery periods. A terminal blood sample (2 ml) was collected by cardiac puncture for determination of osmolality.
2.3. Glomerular filtration rate measurements Glomerular filtration rate (GFR) was determined from the clearance of inulin in the same animals prepared for renal studies. All animals were given a priming dose (0.3 Ci in 0.3 ml saline) of 3[H] inulin (Amersham, Buckinghamshire, UK; specific activity 1.74 Ci.mmol⫺1) and then placed on a continuous intravenous infusion at 150 l.min⫺1 of 0.077 M NaCl containing inulin (0.14 Ci.min⫺1) throughout the experimental period. Blood samples (200 l) were taken at 1-h intervals into heparinized haematocrit tubes throughout the 4-h postequilibration period for measurement of haematocrit before analysis of separated plasma. Aliquots of urine (10 l) and separated plasma (10 l) were counted on a Minaxi  Tri-Carb 4000CA series liquid Scintillation Counter (Packard Instrument Co., Downer’s Grove, IL). 2.4. Analytical methods 2.4.1. Measurement of electrolytes and osmolality Urine volume was determined gravimetrically. Na⫹ and K⫹ were determined by Flame Photometry (Corning model 435 Flame Photometer; Corning Limited, Halstead, UK). The fractional excretion rates of sodium ⫹ ) were calculated. Osmolalities of urine and plasma (FENa were measured on 100-l samples by freezing point depression using a Camlab osmometer (Cambridge, UK). 2.4.2. Plasma ethanol, chloroquine, and hormone measurements Blood was collected from parallel groups of animals prepared for renal studies by cardiac puncture in cooled heparinized containers at the end of 1 h 20 min treatment or corresponding time for control animals. Blood was placed and spun in a Sorvall RT6000 Refrigerated Centrifuge (Dupont, Newtown, MA) at 2,500 g.min⫺1 for 10 min at ⫺4⬚C, and separated plasma was stored at ⫺20⬚C until measurement of aldosterone and AVP. Measurements for ethanol and chloroquine were performed by 12 h after separation. Plasma ethanol was measured by gas chromatography. Chloroquine concentration was assayed after extraction. Initially, 0.5 ml of potassium hydroxide (16 M) was added to a separator flask containing 4 ml plasma, followed by 25 ml chloroform. The mixture was well shaken. Subsequently, chloroquine was extracted from the lower chloroform layer (21 ml) with 4 ml hydrochloric acid (1 M) and measured by UV Spectroscopy (Shimadzu U-V-Visible160A) at 343 nm spectrophotometrically using plasma as a blank. Plasma aldosterone concentration was measured by Coat-A-Count procedure using a kit from Diagnostic Products Corporation (Los Angeles, CA). This is a solid-phase radioimmunoassay procedure based on aldosterone-specific antibody immobilized to the wall of a polypropylene tube. The lower limit of detection was
C.T. Musabayane et al. / General Pharmacology 34 (2000) 43–51
44 fmol.l⫺1. Inter- and intra-assay coefficients of variation were 8.1% (n ⫽ 20) and 8.3% (n ⫽ 20), respectively. AVP was determined as described by Forsling and Peysner (1988) using the DSL-1800 Arginine Vasopressin Radioimmunoassay kit from Diagnostic Systems Laboratories (Webster, TX). Vasopressin was extracted from plasma using Sep Pak C18 cartridges (Millipore Water Associates, Harrow, UK). The lower limit of detection was 0.5 fmol.l⫺1 and intra- and interassay variations were 7.7% (n ⫽ 12) and 11.9% (n ⫽ 12), respectively. 2.4.3. Data presentation and statistical analysis Values are presented as means ⫾ SEM. Data in control rats were used as a baseline for comparison. Renal electrolyte excretion and urine flow rate are presented graphically showing 20-min collections over the 4-h postequilibration period. The total fluid voided, urine osmolalities, and Na⫹ and K⫹ excreted during the 1 h 20 min of chloroquine and/or ethanol administration were compared to that of control animals at the corresponding time. The plasma concentrations of ethanol, chloroquine, and hormones data from treated animals were compared to control values to evaluate the effects of each treatment. All data were subjected to one-way
45
analysis of variance and Scheffe’s multiple comparison was used to resolve any apparent differences. A value of p ⬍ 0.05 was considered significant. 3. Results 3.1. Urinary effects of chloroquine and/or ethanol The urine flow and Na⫹ excretion rates in vehicleinfused control animals during the 4-h postequilibration period stabilized at rates that closely approximated the rates of infusion (Fig. 1, A and B). The urine flow rate ranged from 141 to 163 l.min⫺1 compared with the infusion rate of 150 l.min⫺1. Na⫹ excretion rates stayed around 9 mol.min⫺1, a value that was not significantly different from the infusion rate (11.6 mol.min⫺1). K⫹ excretion rates were stable, approximating 3.5 mol.min⫺1 throughout the postequilibration period. Administration of chloroquine for 1 h 20 min did not affect urine flow rate (Fig. 1C). However, it did affect Na⫹ excretion rate, which increased to 13.1 ⫾ 0.9 mol.min⫺1 (n ⫽ 8) 20 min after the commencement of chloroquine administration, a value that was significantly (p ⬍ 0.01) higher than that of controls (9.1 ⫾ 0.4 mol.min⫺1, n ⫽ 8) for the corresponding period (cf.
Fig. 1. Urine flow and Na⫹ and K⫹ excretion rates in 0.077 M NaCl infused control rats (A and B) and rats administered chloroquine (Chq) at 0.06 g.min⫺1 (C and D) for 1 h 20 min. Values are presented as means for each 20-min collection for the 4-h postequilibration period; vertical bars indicate SE of means.
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Fig. 2. The urine flow, Na⫹ and K⫹ excretion rates in rats administered ethanol (EtoH) at dose rates at either 2.4 g.min⫺1 (A and B) or 24 g.min⫺1 (C and D). Values are presented for each 20-min collection for the 4-h postequilibration period; vertical bars indicate SE of means.
Fig. 1, B and D). K⫹ excretion rates were not altered by this treatment. In comparison to control animals, ethanol administration at both dose rates (2.4 or 24 g.min⫺1) reduced urine flow rate, resulting in less volume of urine voided during treatment period (See Fig. 2 and Table 1). The reduction in urine flow rate was maintained during the recovery period in animals treated with the higher dose (cf. Fig. 2, A and C). Ethanol infused at the low dose rate increased urinary Na⫹ excretion rates and the total amount excreted, but the total amount of K⫹ excreted was reduced (Table 1). Ethanol at 24 g.min⫺1 increased Na⫹ excretion rate to 15.51 ⫾ 0.68 mol.min⫺1 by 20
min, but the rate subsequently dropped throughout the treatment period to reach 6.94 ⫾ 0.86 mol.min⫺1 by the last treatment. The total amount of Na⫹ excreted during the treatment compared to that excreted by control animals at the corresponding time (Table 1), while urinary K⫹ output was reduced. The administration of combined chloroquine and ethanol at a lower dose (2.4 g.min⫺1) decreased diuresis, which reached 87 l.min⫺1 20 min after the cessation of ethanol infusion (Fig. 3A). The total volume of fluid voided during the treatment period was significantly less than that of control animals (see Table 1). However, combined administration of chloroquine and etha-
Table 1 Comparison of terminal plasma osmolalities, total amounts of urine voided, urine osmolalities, Na⫹ and K⫹ excreted during the 1 h 20 min treatment period and in groups of animals administered chloroquine and/or ethanol with control rats Treatment
UV (ml)
Control Chq (0.06 g.min⫺1) EtoH (2.4 g.min⫺1) EtoH (24 g.min⫺1) Chq ⫹ EtoH (2.4 g.min⫺1) Chq ⫹ EtoH (24 g.min⫺1)
11.82 11.59 8.55 6.08 7.46 11.20
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.03 1.16 0.33* 0.19* 0.14* 0.49
Na⫹ (mol) 842 1054 1140 817 883 839
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
K⫹ (mol) 51 44* 22* 39 49 19
* p ⬍ 0.01 by comparison with control animals. n ⫽ 8 in all groups.
275 265 153 176 149 191
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
20 22 10* 17* 26* 35*
Urine osmolality (mOsm.kg⫺1 H2O) 285 274 161 152 160 265
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
21 25 14* 17* 10* 23
Plasma osmolality (mOsm.kg⫺1 H2O) 279 284 263 228 255 273
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
9 5 3* 3* 6* 9
C.T. Musabayane et al. / General Pharmacology 34 (2000) 43–51
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Fig. 3. The urine flow, Na⫹ and K⫹ excretion rates in rats simultaneously administered chloroquine (Chq) at 0.06 g.min⫺1 and ethanol (EtoH) at either 2.4 g.min⫺1 (A and B) or 24 g.min⫺1 (C and D). Values are presented for each 20-min collection for the 4-h postequilibration period; vertical bars indicate SE of means.
nol at a higher dose (24 g.min⫺1) was associated with temporary diuresis, which reached 114 l.min⫺1 by 40 min after the start of infusion before rising to values that did not statistically differ from control animals at corresponding times (Fig. 3C). The total volume of fluid voided during the treatment period did not significantly differ from values observed in control animals at the corresponding time. Na⫹ excretion rates were not significantly altered by the administration of chloroquine and ethanol (at all doses) when compared to control, while K⫹ excretion was reduced (Fig. 3, B and D). 3.2. Urine osmolality and FENa⫹ rate during the treatment period and terminal plasma osmolality The mean FENa⫹ rate during chloroquine infusion was not significantly altered when compared to values of control rats at the corresponding time (Fig. 4C). However, ethanol alone at a low dose significantly (p ⬍ 0.05) increased the mean FENa⫹ rate (Fig. 4C). Combined chloroquine and ethanol at all doses did not significantly change the mean FENa⫹ rate. The urine osmolalities during the administration of chloroquine alone or in combination with ethanol at a high dose were not significantly different from that of control animals at the corresponding time (Table 1). However,
urine osmolalities in animals administered ethanol alone at both doses or ethanol at a low dose in combination with chloroquine were significantly (p ⬍ 0.01) low by comparison with control rats. The mean differences in FENa⫹ rates and urine osmolalities in the 1 h 20 min of chloroquine and/or ethanol administration were reflected in plasma osmolalities for samples collected at the end of the experiment with animals administered ethanol alone or chloroquine with ethanol at a low dose exhibiting low values (Table 1). 3.3. Plasma hormones, ethanol and chloroquine concentrations, mean arterial blood pressure, and GFR Measurements of haematocrit were relatively stable in all groups of animals, remaining at approximately 42% throughout the 4-h postequilibration period. Fig. 4 shows that the mean arterial blood pressure was significantly (p ⬍ 0.01) elevated above the pretreatment values by acute ethanol infusion at either 2.4 g.min⫺1 (from 129 ⫾ 2 mmHg to 137 ⫾ 1 mmHg) or 24 g.min⫺1 (from 126 ⫾ 1 mmHg to 132 ⫾ 1 mmHg). The increases were associated with an increase in GFR to approximately 4.0 ml.min⫺1, a value significantly higher than that observed during the pretreatment (approximately 3.0 ml.min⫺1). Infusion of chloroquine alone or in com-
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C.T. Musabayane et al. / General Pharmacology 34 (2000) 43–51
Fig. 4. Mean arterial blood pressure (A), GFR (B), and FeNa⫹ (C) rates in control rats and rats administered chloroquine and/or ethanol (EtoH). Values are calculated to indicate 1-h control (pretreatment), 1 h 20 min of treatment, and 1 h 40 min of recovery for the 4-h postequilibration period; vertical bars indicate SE of means.
bination with ethanol did not have any significant effects on either mean arterial blood pressure or GFR. Table 2 shows that chloroquine infusion for 1 h 20 min significantly (p ⬍ 0.01) increased plasma AVP concentrations by comparison with control animals. The AVP levels were further elevated when chloroquine was co-administered with ethanol in an ethanol dosedependent manner. By comparison with animals administered chloroquine alone, plasma chloroquine concentrations were increased between 97 and 167% following 1 h 20 min of combined chloroquine and ethanol at both doses. Plasma ethanol concentrations were not detectable after 1 h 20 min administration of chloroquine and/ or ethanol at the low dose administration rate (2.4 g.min⫺1). Simultaneous chloroquine and ethanol infusion significantly (p ⬍ 0.01) increased plasma chloroquine concentrations in an ethanol dose-depen-
dent manner by comparison with animals administered chloroquine alone. Plasma ethanol concentrations were detectable in groups of rats administered ethanol at 24 g.min⫺1 alone or in combination with chloroquine. The mean plasma ethanol concentrations following simultaneous infusion of chloroquine and ethanol at a high dose were elevated by 26% by comparison with that of ethanol alone. In contrast, plasma ethanol concentrations were not detectable in animals following infusion of ethanol alone at a low dose or in combination with chloroquine. 4. Discussion The results of the current study extend our previous observations that acute chloroquine administration increases plasma AVP concentrations (Musabayane et
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Table 2 Plasma chloroquine, ethanol, aldosterone, and AVP concentrations in control rats and in animals administered chloroquine and/or ethanol for 1 h 20 min
Control Chq (0.6 g.min⫺1) EtoH (2.4 g min⫺1) EtoH (24 g min⫺1) Chq ⫹ EtoH (2.4 g min⫺1) Chq ⫹ EtoH (24 g min⫺1)
Ethanol (mg.dl⫺1)
Chloroquine (g. ml⫺1)
Aldosterone (nmol.l⫺1)
not detected not detected not detected 39.32 ⫾ 3.90 not detected 49.60 ⫾ 3.35
not measured 2.89 ⫾ 0.22 not measured not measured 5.70 ⫾ 0.43* 7.71 ⫾ 0.83*,**
1.86 1.77 1.83 1.79 1.77 2.23
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.20 0.19 0.09 0.29 0.29 0.24*
AVP (fmol.l⫺1) 9.73 15.65 18.46 29.70 17.29 33.87
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
1.64 2.49* 3.42* 4.32* 4.21* 6.18*
* p ⬍ 0.01 by comparison with control animals. ** p ⬍ 0.01 by comparison with the group administered Chq ⫹ EtoH (2.4 g min⫺1). n ⫽ 8 in all groups.
al., 1996) by showing that this effect is further enhanced when the antimalarial is co-administered with ethanol. The dose rate for intravenous chloroquine infusion resulting in total dose of 16 g.kg⫺1 was significantly (p ⬍ 0.01) lower than doses previously used in rats (600–1500 g.kg⫺1; Adelusi et al., 1982) and humans (750–850 g.kg⫺1; White et al., 1988). The administration dose rates of ethanol, which resulted in total doses of 0.64 or 6.4 mg.kg⫺1, were much lower than doses previously used in rats (1.2–2.0 g.kg⫺1 Wu et al., 1992). Ethanol administration caused dose-dependent increases in plasma AVP concentrations and responses in renal Na⫹ handling. The low dose increased the total amounts of urinary Na⫹ loss, but the high dose did not significantly affect the total urinary Na⫹ loss despite eliciting high excretion rates in the initial 20 min (see Fig. 2D). We suggest adaptive changes in response to the initial elevated urinary Na⫹ loss led to subsequent Na⫹ retention by 40 min of treatment. This possibly contributed to the differences in the total amounts of urinary Na⫹ after infusion of ethanol at either low or high dose rate. Dose-dependent effects of ethanol on (Na⫹-K⫹)-ATPase activity, an enzyme that increases Na⫹ transport in renal collecting ducts, may also be invoked to explain the differences. For instance Rodrigo et al. (1998) have reported ethanol dose-dependent effects on the (Na⫹-K⫹)-ATPase. Indeed, we have reported Na⫹ retention after administration of ethanol at 60 g.kg⫺1 (Musabayane et al., 1985). Chloroquine and/ or ethanol administration led to significant reduction in urinary K⫹ loss. The hormonal basis, if any, of this retention remains unresolved. Previously, we attributed urinary Na⫹ loss after acute chloroquine infusion to elevated plasma AVP levels (Musabayane et al., 1993). However, combined administration of chloroquine and ethanol at both doses did not affect renal Na⫹ excretion despite inducing significant increases in plasma AVP concentrations (Table 2). This suggests the ethanol inhibition of the chloroquine-induced renal Na⫹ loss cannot be ascribed to ethanol influencing plasma AVP concentrations. Presumably, ethanol induced alteration in membrane fluidity (Villanueva et al., 1994), and direct interference with
functions of transport proteins in renal brush border (Deves and Kripka, 1990) altered the renal effects of chloroquine. We cannot exclude the influence of chloroquine or ethanol on the autonomic nervous system by a direct action on tubular reabsorption mechanisms or an indirect action on renal haemodynamics to affect renal electrolyte excretion. For instance the reported stimulation of the sympathetic nervous system by ethanol (Delarue et al., 1997) can increase blood pressure and, if this is coupled with a rise in GFR, can increase renal electrolyte excretion. Indeed, ethanol alone increased mean arterial blood pressure by 30%, and this was associated with an increase in GFR by comparison with control animals. However, chloroquine has an antimuscarinic effect (Lot and Bennet, 1982; Mubagwa and Adler, 1988) and as such would be expected to reduce sympathetic responses to cause renal Na⫹ retention. Therefore, renal electrolyte excretion after chloroquine and/or ethanol administration can partly be attributed to the influence of chloroquine or ethanol on the sympathetic nervous system. Acute ethanol at both doses resulted in antidiuresis, perhaps because of high plasma AVP levels. However, some studies suggest that ethanol ingestion acutely alters water balance with increased diuresis caused by inhibition of AVP release (Eisenhofer and Johnson, 1982). The absence of antidiuresis despite increases in plasma AVP concentrations after the administration of chloroquine alone or with ethanol is perhaps caused by inhibition of cAMP production by chloroquine. We have observed in isolated rat inner medullary collecting ducts that chloroquine inhibits the AVP-induced cAMP production that mediates the antidiuretic effects of AVP (Musabayane et al., 1997). The plasma ethanol concentrations after infusion of a lower dose of ethanol alone or in combination with chloroquine were undetectable, but were observed after infusion of ethanol at high doses alone or in combination with chloroquine. Perhaps the ability of hepatic catalase to rapidly metabolize low ethanol concentration (Lieber, 1997) accounted for these differences. Plasma chloroquine and ethanol concentrations were
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significantly elevated in animals when chloroquine and ethanol were simultaneously administered, by comparison with groups of animals administered either chloroquine alone or ethanol alone. This suggests reduction in chloroquine or ethanol metabolism following coadministration of these compounds. Presumably chloroquine and ethanol compete for a partially common detoxification process, which includes binding with cytochrome P450. The cytochrome P450 system involves not only the ethanol oxidizing system (Lieber, 1997), but also the detoxification process of chloroquine (Thabrew and Ioannides, 1984). This would be of clinical importance if this was established because simultaneous intake of chloroquine and ethanol in areas where malaria is endemic may exacerbate the reported impairment of renal electrolyte handling associated with chloroquine administration in the rat (Musabayane et al., 1996) and man (Musabayane et al., 1999). Since we have shown no significant differences in renal function between anesthetized and conscious salineinfused preparations (Balment et al., 1984; Brimble et al., 1986), it is possible that the effects of chloroquine and/or ethanol on renal electrolyte and fluid handling seen in the present study may apply to awake conditions. In summary, results of the present study confirm that the pathophysiology of renal regulation of water and electrolytes after chloroquine and/or ethanol administration may be commonly ascribed to altered plasma AVP concentrations, but may also be caused by other intrarenal effects such as alterations in GFR. The results also suggest dose dependent responses on renal electrolyte excretion in response to ethanol.
Acknowledgments This study was funded by the University of Zimbabwe Research Board (YYH010/3580) and The Kapnek Charitable Trust. The authors thank Byk Gulden, Konstanz, for the gift of Trapanal.
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