Sodium valproate stimulates potassium and chloride urinary excretion in rats: gender differences

Background The diuretic effect of valproates and its relation to urinary potassium (K+) and chloride (Cl-) excretion have not yet been investigated, so the aim of this study was to evaluate the influence of a single dose of sodium valproate (NaVPA) on 24-h urinary K+ and Cl- excretion in young adult Wistar rats of both genders. For measurement of K+ in urine, the same animals and samples as in our earlier publication were used (Pharmacology 2005 Nov, 75:111–115). The authors propose a new approach to the pathophysiological mechanisms of NaVPA effect on K+ and Cl- metabolism. Twenty six Wistar rats were examined after a single intragastric administration of 300 mg/kg NaVPA (13 NaVPA-male and 13 NaVPA-female), 28 control intact Wistar rats (14 males and 14 females) were studied as a control group. The 24-h urinary K+, Cl-, creatinine and pH levels were measured. Results Total 24-h diuresis and 24-h diuresis per 100 g of body weight were found to be significantly higher in NaVPA-rats of both genders than in rats of the control group (p < 0.05). The data showed NaVPA to enhance 24-h K+ excretion in NaVPA-males and NaVPA-females with significant gender-related differences: 24-h K+ excretion in NaVPA-male rats was significantly higher than in control males (p = 0.003) and NaVPA-female rats (p < 0.001). Regarding the 24-h K+ excretion, NaVPA-female rats did not show a statistically significant difference versus females of the control group (p > 0.05). 24-h urinary K+ excretion per 100 g of body weight in NaVPA-male rats was significantly higher than in control males (p = 0.025). NaVPA enhanced Cl- urinary excretion: 24-h Cl- urinary excretion, 24-h urinary Cl- excretion per 100 g of body weight and the Cl-/creatinine ratio were significantly higher in NaVPA-male and NaVPA-female rats than in gender-matched controls (p < 0.05). 24-h chloriduretic response to NaVPA in male rats was significantly higher than in female rats (p < 0.05). Conclusion NaVPA causes kaliuretic and chloriduretic effects with gender-related differences in rats. Further investigations are necessary to elucidate the mechanism of such pharmacological effects of NaVPA.


Background
Currently there are experimental data that valproate (branched-chain fatty acid, valproic acid) increases the turnover of γ-aminobutyric acid (GABA) and thereby potentiates GABAergic functions [1]. The specificity of valproate for GABA suggests that this interaction may be an important mechanism through which sodium valproate (NaVPA) exerts its pharmacological effects [2]. Recently NaVPA has shown to enhance the urinary excretion of sodium (Na + ) and chloride (Cl -) ions in both genders, but the 24-h chloriduretic response in male rats to NaVPA was significantly higher than in female rats [3]. The effect of NaVPA on potassium ion (K + ) excretion was not yet studied.
The aim of the present study was to evaluate the effect of NaVPA on urininary K + and Clexcretion in Wistar rats of both genders and to discuss the NaVPA effects on K + and Clmetabolism that could be related to NaVPA pharmacological properties.
The GABA type A receptor (GABA(A)) is an ionotropic receptor. Its subunits form a functional Clchannel [4,5]. The GABA(A) receptor subunits are expressed in Wistar rat kidney proximal convoluted and straight tubules [6]. The GABA(A) receptor is rapidly activated by valproate in the brain [7]. Clchannels play a critical role in the functioning of the nervous system by asserting control over voltage potentials across the plasma membrane [8]. There are gender-related differences in Cltransport across the cell membrane, intracellular Cllevel and the sensitivity of Cltransport to vasopressin in smooth muscle cells of rats [9]. Intracellular Cllevel and Cltransport differences could be important in the regulation of cellular processes and could help to explain certain functional differences of cells [9,10]. Clis an important factor of intracellular pH [11], which is involved in the complex of cell function regulation.
Investigations show that K + -Clcotransport takes part in the regulation of signaling pathways involved in several tissue and cell types from different species [12]. In modeling Cltransport in the rat proximal tubule, Weinstein has found that Clions efflux from the cell predominantly via the K + -Clcotransport mechanism [13]. The intracellular Cllevel is dependent upon the K + -Clco-transporter (KCC) that determines whether neurons respond to GABA by depolarization or hyperpolarization. However, the role of KCC-dependent chloride homeostasis in the regulation of spontaneous activity of neuronal circuits via GABA(A) receptor is still unknown. Findings suggest that KCCdependent chloride homeostasis is mainly involved in GABA(A) receptor-mediated synaptic inhibition [14]. There are no investigational data on the interaction between KCC and GABA receptors, K + homeostasis or NaVPA effects on K + and Cltransport in the kidney.
The study provides data to show that NaVPA in rats, along with the known diuretic and chloriduretic effects, causes also a kaliuretic effect that has not yet been investigated. For measurement of K + in urine, the same animals and samples as in our earlier publication were used (Pharmacology 2005 Nov, 75:111-115).

Methods
Twenty-six Wistar rats (13 NaVPA-males and 13 NaVPAfemales) were examined after a single intragastric dose of 300 mg/kg sodium valproate (Convulex, 300 mg/ml, drops (water solution, pH 9.0), Gerot Pharmazeutika Wien, Austria). In addition, 28 Wistar intact rats (14 males and 14 females) were examined as a control group. NaVPA dosage was chosen in accordance with data of preclinical pharmacodynamic studies of NaVPA [15]. The study was approved by the Lithuanian Committee for Animals Care and Use (No. 0019; 2005). The mean age of control rats was 91 ± 9 days for males and 90 ± 8 days for females, and the mean age of NaVPA-rats was 97 ± 10 days for males and 95 ± 9 days for females. The mean weight of male rats was 283 ± 30 g in control group and 298 ± 23 g in NaVPA-rats. The mean weight of female rats was 236 ± 18 g in control and 240 ± 16 g in NaVPA-rats. The weight was significantly higher in male than in female rats in both groups (p < 0.05).
The animals were housed in standard colony cages with free access to food (chow pellets) and tap water. The room temperature was 21 ± 1°C. The rats were on a natural light-dark cycle. All experiments were performed according to the institutional guidelines for animal care in order to avoid any unnecessary distress to the animals and to reduce the number of animals used. The animals were housed in described conditions and acclimated for at least 5 days before experiments. 24-h urine was collected keeping a rat alone in a special cage (diuresis cage for rats 3700D000/3701D000, Tecniplast, Italy) for 24 h (from 9:00 a.m. till 9:00 a.m. of the next day) with free access to tap water, without food, in the same temperature and light conditions. 24-h urine was collected after a single dose administration. 24-h urinary K + , Cllevels were analyzed with an EML-105 electrolyte analyzer (Radiometer, Denmark). Urinary pH levels were measured with a pH/mV/ion meter (ION Meter pH 340/ION, Germany).
We calculated the 24-h excretion of K + , Cl -, creatinine, K + / creatinine, Cl -/creatinine ratio, as well as 24-h diuresis and 24-h urinary K + , Clexcretion per 100 g of body weight. Data were expressed as mean ± SD values from n animals. Comparisons between the groups were carried out using Student' s t test. A value of p < 0.05 was considered significant. Correlations between two variables were investigated by the method of linear correlation analysis. The Pearson correlation coefficient r, which represents the linear relationship between two variables, was applied; a value of p < 0.05 was considered significant. STATISTICA for Windows software (StatSoft, USA, 1995) was used to perform the analysis of our data.

K + excretion in control and NaVPA-rats
No statistically significant gender-related differences in 24-h urine K + excretion and in the K + /creatinine ratio were determined in control rats (p > 0.05). 24-h urinary K + excretion per 100 g of body weight was significantly higher in control females than in control males (p = 0.04; Table 1). 24-h K + excretion in NaVPA-male rats was significantly higher than in control male (p = 0.003) and NaVPA-female rats (p < 0.001). 24-h K + excretion in NaVPA-female rats did not show statistically significant difference versus female controls (p > 0.05). 24-h urinary K + excretion per 100 g of body weight in NaVPA-male rats was significantly higher than in control males (p = 0.025; Table 1). No gender-related differences in this index were found in NaVPA-rats. This index in NaVPA-female rats did not show any statistically significant difference as compared with female controls (p > 0.05).
The K + /Clratio (Table 2) in NaVPA-males and NaVPAfemales was significantly lower as compared to control males (p < 0.0005) and control females (p < 0.0005), without any statistically significant gender-related differences.

Clexcretion in control and NaVPA-rats
No significant gender differences of 24-h urine, Clexcretion and Clexcretion per 100 g of body weight and Cl -/ creatinine ratio were determined in control rats, either (p > 0.05; Table 2).
Compared to control, 24-h urinary Cllevels were significantly (p < 0.05) higher in both genders of experimental animals. 24-h urinary Clexcretion, 24-h urinary Clexcretion per 100 g of body weight and the Cl -/creatinine ratio in both sexes of NaVPA-rats were significantly higher than in gender-matched controls (p < 0.001). Chloride excretion (24-h urinary Clexcretion, 24-h urinary Clexcretion per 100 g of body weight and the Cl -/creatinine ratio) was found significantly higher in NaVPA-male than in NaVPAfemale rats (p < 0.05; Table 2). 24-h creatinine excretion in NaVPA-male rats (0.110 ± 0.025 mmol) was significantly higher than in NaVPA-female rats (0.082 ± 0.017 mmol; p < 0.005). No gender-related difference in 24-h creatinine excretion between control male and female rats was determined (p > 0.05).
The correlation between 24-h urinary K + and Clexcretion and between respective electrolyte and 24-h urine pH in control male and female rats was not statistically significant (p > 0.05). The correlation coefficient of 24-h urinary K + excretion and 24-h urinary Clexcretion statistically significantly differed (p = 0.03) only between control males (r = -0.28) and control females (r = 0.49).

Discussion
Cland K + fluxes play a crucial role in synaptic inhibition, cell pH regulation, as well as in cell volume control and tissue susceptibility to seizures [16,17]. A recent study showed that NaVPA, alongside the diuretic effect, enhances sodium and Clexcretion with urine [3]. Acute and subacute administration of valproic acid has been shown to exert a moderate diuretic effect on rats [9,18,19].
The study data showed that intragastric 300 mg/kg NaVPA significantly increased 24-h K + excretion in the urine of NaVPA-male rats. 24-h K + excretion in NaVPA-male rats was significantly higher than in control males and NaVPA-female rats. 24-h urinary K + excretion per 100 g of body weight in NaVPA-male rats was significantly higher than in control male rats.
Correlation between urine pH and Clexcretion in NaVPA male and female rats Figure 3 Correlation between urine pH and Clexcretion in NaVPA male and female rats. * -Statistically significant correlation.
Correlation between 24-h diuresis and K + excretion in NaVPA male and female rats Figure 1 Correlation between 24-h diuresis and K + excretion in NaVPA male and female rats. * -Statistically significant correlation.
Correlation between 24-h diuresis and Clexcretion in NaVPA male and female rats Figure 2 Correlation between 24-h diuresis and Clexcretion in NaVPA male and female rats. * -Statistically significant correlation.
The enhanced excretion of K + and Clwith urine and the related gender differences of NaVPA effect could be important for elucidating the pathophysiological phenomena related to NaVPA pharmacology. GABA functions appear to be triggered by GABA binding to its ionotropic receptors, which are ligand-gated Clchannels [4,5]. The Clchannel of the GABA(A) receptor is activated by valproic acid in brain cells [7]. GABA was also found to activate K + conductance in the central nervous system [20], it is involved in a wide variety of physiological functions in tissues and organs outside the brain [12,21]. Activation of the GABA(A) receptor leads to a stimulation of Na + -K + -2Clcotransporter in brain cells, and this results in a loss of intracellular Cl - [22] or in an upregulation of KCC which is important in maintaining the low intracellular Cllevel [23].
Studies demonstrating K + -Clcotransport in rabbit proximal tubules have shown a coupled K + -Clmovement from the cell to peritubular fluid [24]. In addition, several modes of coupled K + and Clmovement have been shown in K + excretion by renal tubules: directly coupled K + -Clcotransport, parallel K + and Clconductance, parallel K + / H + and Cl -/HCO3exchangers and Na + -K + -2Clcotransport [25]. In modeling Cltransport in the rat proximal tubule, Weinstein found that Cleffluxes from the cell predominantly via K + -Clcotransport [13].
The presence and physiological significance of GABA or GABA(A) receptors in nonneural tissue is less clear [26]. The effect of pharmacological manipulation of GABAergic transmission on KCC activity in the kidney remains to be clarified. GABA immunoreactivity in the rat kidney was predominantly confined to renal tubules, including the ascending parts of the distal tubules, and the loop of Henle, the collecting tubules and ducts, and the connective parts of the convoluted tubules. The high K + concentration evoked an efflux of endogenous GABA from rat kidney slices. GABA released from renal tubular epithelium and transported with urine might be involved in the modulation of K + transport in the urinary tract cells [27]. However, our findings allow to hypothesize that GABA(A) subunits may play a role in basolateral membrane Cltransport. These findings support the suggestion of other investigators, that subunits of the ligand-gated Clchannel superfamily may be involved in renal Clexcretion [6].
NaVPA exerts a gender-related effect on urinary Clexcretion: 24-h urinary Clexcretion was significantly higher in rats of both sexes, but Clexcretion was significantly higher in male than in female rats. In NaVPA-male rats, the negative correlation between Clexcretion and 24-diuresis and between Clexcretion and urine pH was significant, but such correlations were not characteristic of control male or NaVPA-female rats. The K + /Clratio in NaVPA-males and NaVPA-females was significantly lower as compared to control males and control females, without gender-related differences. There are no data that could contribute to elucidating the mechanisms of NaVPA-induced enhanced urinary Clexcretion. It is known that the alkaline extracellular pH increases the GABA(A) channel opening frequency and decreases the duration of the long-closed state in rat hypothalamus [28]. There are no data that NaVPA influences tubular intracellular and extracellular pH. Gender-related differences in intracellular Clconcentration and Cltransport in smooth muscle cells of male and female rats have been reported [9]. Sexual dimorphism in the expression of KCC, GABA receptors and in GABA release in the nervous system of rats is a known fact [29][30][31][32][33][34][35].
Tissue distribution studies with radiolabeled NaVPA in rodents have shown that NaVPA distributed mainly in the extracellular space; high levels of radiolabeled NaVPA were found in the liver and kidney [36]. We failed to find data on gender-related differences of NaVPA pharmacokinetics, pharmacodynamics, or gender differences of NaVPA metabolism in rats. The NaVPA-induced K + and Clexcretion enhancement may be related to the upregulated renal hemodynamics, as the NaVPA doses applied are known to reduce arterial blood pressure in rats: NaVPA provoked a prolonged cardiovascular depression which was very similar to that observed with high i.v. doses of GABA [18,37]. The mechanism of the cardio depressive effect of NaVPA is not clear; it seems not to involve interference with peripheral vascular noradrenergic activity or arterial baroreflex control [38]. Furthermore, Clis an important factor of renal vascular tone or renal hemodynamics regulation. Renal arterial infusion of Clacts as a direct vasoconstrictor [39]; retrograde injection of Cl -containing solution into the distal tubule elicits a decrease in a single nephron glomerular filtration rate, whereas injection of sodium ion containing solution without Cldoes not [40]. In oncotically perfused rat kidneys, in the presence of either high or low Clwith sodium kept constant, low Clresulted in a higher glomerular filtration rate than with high chloride [41]. It is not likely that the NaVPA-induced gender-related K + and Clexcretion is linked to Na + -K + -2Clcotransporter inhibition, because the Na + -K + -2Clcotransporter inhibitor furosemide exerts an opposite gender-related chloriduretic effect in rats [42].

Conclusion
NaVPA, alongside the diuretic effect, enhances K + and Clexcretion with urine in rats of both genders. The mechanism of this different gender-dependent effect is not yet clear. The experimental observations reported above may have potentially important pharmacological implications. Elucidation of NaVPA-induced mechanisms of enhanced K + and Clexcretion could be of value while explaining the pharmacological basis of NaVPA action. Thus, further studies of the mechanisms of NaVPA effects on K + and Cltransport in cells could be important.