Monocarboxylate transporters are involved in nutrient and drug distribution in the kidney. Alterations in renal monocarboxylate transporter expression could lead to changes in nutrient reabsorption, drug reabsorption and renal clearance. Previous studies have been demonstrated that MCTs and SMCTs are regulated in a tissue specific manner by sex hormones (22, 23, 25); however, to our knowledge no studies have investigated the influence of the estrus cycle and sex hormones on GHB toxicokinetics and renal MCT/SMCT expression. Here, we demonstrate that GHB toxicokinetics vary over the estrus cycle in females with lower renal clearance in OVX females, as compared to proestrus females. GHB renal clearance in OVX females was similar to that in CST and intact males suggesting that female sex hormones contribute to the observed differences in toxicokinetics. Further, we demonstrated that renal MCT1 membrane expression varies over the estrus cycle, with the lowest expression observed in proestrus females, which is consistent with the observed changes in GHB renal clearance.
GHB demonstrates nonlinear toxicokinetics following iv bolus administration due to capacity-limited metabolism and renal reabsorption (7, 31). In overdose, GHB metabolic clearance is saturated, and renal clearance becomes a significant route of elimination. The toxicokinetic profiles in the present study are consistent with previous studies reporting GHB toxicokinetics in male rats (2, 4, 32, 33). The concentrations which lead to death in humans (post-mortem blood levels: mean 660 mg/L) are lower than those that cause death in rats; however, the concentrations in the current study are in the range of those seen in human overdose (34). Our results demonstrate similar clearance parameters in males as compared to previous literature (33, 35). In the present study, proestrus females demonstrated increased renal clearance and reduced plasma concentrations and exposure (AUC) at 600 mg/kg compared to other cycling females. However, at a dose of 1000 mg/kg there were no differences between cycling females suggesting saturation of GHB clearance and/or distribution pathways. Males and OVX females had lower renal and metabolic clearance at 1000 mg/kg indicating that female sex hormones reduce GHB exposure. Future studies will evaluate higher doses of GHB (up to 1500 mg/kg) and evaluate the impact of individual sex hormones on GHB toxicokinetics and GHB-induced fatality.
Sex hormone-mediated regulation of MCTs was demonstrated in Sertoli cells, skeletal muscle and brain (22, 23, 36); however, there is minimal data in tissues involved in drug distribution. In Sertoli cells, the MCT 4 mRNA level significantly decreased with 17β-oestradiol and dihydrotestosterone treatment (23). Administration of testosterone increased MCT1 and MCT4 protein expression in rat skeletal muscle (22). Oestrogen-related receptor alpha (ERRα) expression can modulate the expression of MCT1 in mice, and the induction of estrogen receptor β (ERβ) expression resulted in increased expression of MCT4 in mesothelioma cells of mice (37). The absence of female sex hormones following ovariectomy induced a decrease in brain MCT2 expression in mice (36). We demonstrated that MCT1 mRNA was significantly higher in male rats compared to CST males and all female groups (Fig. 2A and 2B). This indicates that androgens might plays a role in the increased MCT1 mRNA expression. In contrast, whole cell and membrane MCT1 protein expression was higher in CST males compared to intact males suggesting androgens differentially regulate both transcription and translation. CD147 membrane expression demonstrated significant differences between cycling females, intact males and CST males with the highest expression observed in CST males. Proestrus females had higher CD147 membrane expression compared to other cycling females; however, expression was still less than 50% of that observed in CST males. These expression differences suggest that androgens negatively regulate CD147 membrane localization. MCT1 membrane expression had a similar pattern to membrane CD147 expression suggesting that CD147 contributes to MCT1 membrane trafficking. Taken together these results suggest that female and male sex hormones regulate monocarboxylate transporters expression and membrane trafficking; however, the influence of sex hormones is tissue-specific.
Previous studies in our laboratory have shown that MCT1 and MCT4 are regulated in a sex and sex hormone-dependent manner in the liver with lower MCT1 and MCT4 membrane expression in proestrus females, as compared to males and OVX females (25). In the present study, we demonstrated lower MCT1 membrane expression in proestrus females compared to OVX females, and intact and CST males, which are consistent with our observed renal clearance changes. Renal clearance in proestrus females is higher than that in CST and intact males, due to lower active renal reabsorption clearance. No differences in GHB renal clearance were observed between intact and CST males despite CST males demonstrating higher renal MCT1 membrane expression. Additionally, no differences were observed in SMCT1 expression between males and females. There are two potential scenarios that may explain the observed results: [1] MCT1 membrane expression does not directly correlate with MCT1 activity; and [2] additional transporters are involved in renal reabsorption of GHB.
MCT1 activity at the plasma membrane is influenced by intra- and extra-cellular carbonic anhydrases (38). Intracellular carbonic anhydrase II was demonstrated to increase MCT1 activity without a corresponding increase in MCT1 membrane expression. Extracellular carbonic anhydrases IV and IX were demonstrated to augment the activity of MCT1 (39–42). Additionally, expression of carbonic anhydrase-related proteins VIII, X and XI in Xenopus oocytes were demonstrated to increase the activity of co-expressed MCT1 (43). Sex- or sex hormone-dependent regulation of these proteins may contribute to differences in MCT1 activity in the kidney. Further studies are needed to elucidate the role of sex and sex hormones on the contribution of carbonic anhydrases to MCT1 membrane activity.
Inhibition of MCTs/SMCTs is a proposed treatment strategy for GHB overdose by inhibiting its active renal reabsorption mediated by MCTs and SMCTs (7). Co-administration of MCT substrates and inhibitors, including lactate, increases GHB renal and total clearance in rats (17, 35, 44, 45). Similarly, SMCT1 is involved in renal reabsorption of lactate and pyruvate, which also function as SMCT1 inhibitors (10, 16, 46); however, to date no specific SMCT1 inhibitor has been developed (10). The observed decreases in GHB renal clearance with lactate administration are likely due to combined inhibition of MCTs and SMCTs. After luteolin treatment, the renal and total clearances of GHB are significantly increased because of inhibition of the MCT1-mediated renal reabsorption of GHB (47). As well, the immunosuppressive compounds AR-C117977 and AR-C122982 are potent MCT1 inhibitors (48), and co-administration with GHB lead to increased GHB renal and total clearance (49). However, GHB renal clearance remains less than filtration clearance in the presence of MCT/SMCT inhibitors suggesting that other transporters may contribute to active renal reabsorption of GHB. Further studies are necessary to elucidate the underlying sex and sex hormone-mediated differences in MCT/SMCT transport kinetics and the potential involvement of additional transporters in GHB renal reabsorption.
In summary, we have demonstrated the GHB toxicokinetics and renal clearance vary between sexes, over the estrus cycle in females and in the absence of female sex hormones consistent with the renal expression of MCT1. Our results suggest that females may be less susceptible to GHB-induced toxicity due to decreased exposure secondary to increased renal and metabolic clearance. Future studies should evaluate the sex hormone dependent regulation of enzymes involved in GHB metabolism. Further studies are needed to comprehensively evaluate the contribution of individual sex hormones to differences in GHB toxicokinetics, monocarboxylate transporter expression, and the potential involvement of additional transporters in GHB renal reabsorption.