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

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 Cl^- excretion in Wistar rats of both genders and to discuss the NaVPA effects on K⁺ and Cl^- metabolism that could be related to NaVPA pharmacological properties.

The GABA type A receptor (GABA(A)) is an ionotropic receptor. Its subunits form a functional Cl^- channel [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]. Cl^- channels 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 Cl^- transport across the cell membrane, intracellular Cl^- level and the sensitivity of Cl^- transport to vasopressin in smooth muscle cells of rats [9]. Intracellular Cl^- level and Cl^- transport differences could be important in the regulation of cellular processes and could help to explain certain functional differences of cells [9, 10]. Cl^- is an important factor of intracellular pH [11], which is involved in the complex of cell function regulation.

Investigations show that K⁺-Cl^- cotransport takes part in the regulation of signaling pathways involved in several tissue and cell types from different species [12]. In modeling Cl^- transport in the rat proximal tubule, Weinstein has found that Cl^- ions efflux from the cell predominantly via the K⁺-Cl^- cotransport mechanism [13]. The intracellular Cl^- level is dependent upon the K⁺-Cl^-co-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 KCC-dependent 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 Cl^- transport 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).

Twenty-six Wistar rats (13 NaVPA-males and 13 NaVPA-females) 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⁺, Cl^- levels 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⁺, Cl^- excretion 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.

Cl^- and 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 Cl^- excretion 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.

The enhanced excretion of K⁺ and Cl^- with 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 Cl^- channels [4, 5]. The Cl^- channel 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⁺-2Cl^- cotransporter 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 Cl^- level [23].

Studies demonstrating K⁺-Cl^- cotransport in rabbit proximal tubules have shown a coupled K⁺-Cl^- movement from the cell to peritubular fluid [24]. In addition, several modes of coupled K⁺ and Cl^- movement have been shown in K⁺ excretion by renal tubules: directly coupled K⁺-Cl^- cotransport, parallel K⁺ and Cl^- conductance, parallel K⁺/H⁺ and Cl^-/HCO3^- exchangers and Na⁺-K⁺-2Cl^- cotransport [25]. In modeling Cl^- transport in the rat proximal tubule, Weinstein found that Cl^- effluxes from the cell predominantly via K⁺-Cl^- cotransport [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 Cl^- transport. These findings support the suggestion of other investigators, that subunits of the ligand-gated Cl^- channel superfamily may be involved in renal Cl^- excretion [6].

NaVPA exerts a gender-related effect on urinary Cl^- excretion: 24-h urinary Cl^- excretion was significantly higher in rats of both sexes, but Cl^- excretion was significantly higher in male than in female rats. In NaVPA-male rats, the negative correlation between Cl^- excretion and 24-diuresis and between Cl^- excretion and urine pH was significant, but such correlations were not characteristic of control male or NaVPA-female rats. The K⁺/Cl^- ratio 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 Cl^- excretion. 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 Cl^- concentration and Cl^- transport 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 [29–35].

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 Cl^- excretion 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, Cl^- is an important factor of renal vascular tone or renal hemodynamics regulation. Renal arterial infusion of Cl^- acts 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 Cl^- does not [40]. In oncotically perfused rat kidneys, in the presence of either high or low Cl^- with sodium kept constant, low Cl^- resulted in a higher glomerular filtration rate than with high chloride [41]. It is not likely that the NaVPA-induced gender-related K⁺ and Cl^- excretion is linked to Na⁺-K⁺-2Cl^- cotransporter inhibition, because the Na⁺-K⁺-2Cl^- cotransporter inhibitor furosemide exerts an opposite gender-related chloriduretic effect in rats [42].

NaVPA, alongside the diuretic effect, enhances K⁺ and Cl^- excretion 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 Cl^- excretion could be of value while explaining the pharmacological basis of NaVPA action. Thus, further studies of the mechanisms of NaVPA effects on K⁺ and Cl^- transport in cells could be important.