Analysis of sex difference in the tubular reabsorption of lithium in rats

types of transporters mediate the tubular reabsorption of lithium in rats, and suggested that sodium-dependent phosphate transporters play a role in lithium reabsorption with high affinity. In the present study, we examined sex differences in lithium reabsorption in rats. When lithium chloride was infused at 60 µg/min, creatinine clearance and the renal clearance of lithium were lower, and the plasma concentration of lithium was higher in female rats. These values reflected the higher fractional reabsorption of lithium in female rats. In rats infused with lithium chloride at 6 µg/min, the pharmacokinetic parameters of lithium examined were all similar in both sexes. The fractional reabsorption of lithium was decreased by foscarnet, a representative inhibitor of sodium-dependent phosphate transporters, in male and female rats when lithium chloride was infused at the low rate. Among the candidate transporters mediating lithium reabsorption examined herein, the mRNA expression of only PiT2, a sodium-dependent phosphate transporter, exhibited sexual dimorphism. The present results demonstrated sex differences in the tubular reabsorption of lithium with low affinity in rats.


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Lithium is the gold standard treatment for bipolar disorder (Won and Kim 2017). Its therapeutic range is narrow, and plasma concentration of lithium is clinically monitored.
Clinicians consider steady-state concentrations of between 0.6 and 1.2 mEq/l to be optimal for the maintenance of bipolar disorder and between 0.8 and 1.5 mEq/l to be ideal for the acute management of manic episodes (Finley et al. 1995). After glomerular filtration, 75% of lithium is reabsorbed by renal tubules, mainly the proximal tubules (Finley 2016).
Previously, we intravenously injected lithium chloride at 25 mg/kg and 2.5 mg/kg as a bolus to rats, and showed that at least two types of transporters mediate the tubular reabsorption of lithium in rats: a low-affinity type and a high-affinity type (Uwai et al. 2018). Lithium reabsorption with low and high affinities was inhibited by acetazolamide and foscarnet, a typical inhibitor for sodium-dependent phosphate transporters, respectively (Uwai et al. 2018;Uwai et al., 2020). The molecular mechanisms underlying lithium reabsorption have not yet been elucidated in detail. In the present study, we examined sex differences in the reabsorption of lithium using Wistar/ST rats. The mRNA expression levels of the candidate transporters that mediate lithium reabsorption in the kidney cortex were also compared between both sexes. Animals were treated in accordance with regulations of the Institutional Animal Use and Care Committee of School of Pharmacy, Aichi Gakuin University. Male and female Wistar/ST rats were from Chubu Kagaku Shizai (Nagoya, Japan). Rats were anesthetized with ethyl carbamate and -chloralose. Catheters were inserted into the femoral artery and femoral vein with polyethylene tubes (SP-31; Natsume Seisakusho, Tokyo, Japan) filled with heparin solution (50 IU/mL) for blood sampling and drug administration, respectively.
Urine was collected from the urinary bladder catheterized with SP-31 polyethylene tubes.
Lithium chloride (Wako Pure Chemical Industries, Osaka, Japan) in saline was infused at 60 µg/min or 6 µg/min for 2 h. At the slow infusion of lithium chloride, mannitol (Wako Pure Chemical Industries) was infused at 750 µg/min to achieve a sufficient urine flow.
Sodium foscarnet (Cayman Chemical Company, Ann Arbor, MI, USA) was infused at 600 µg/min to examine the contribution of sodium-dependent phosphate transporters to lithium reabsorption.
Bladder urine samples were collected in the final 20 minutes of the lithium infusion, and blood sampling was performed at its midpoint.
Creatinine and phosphate concentrations in urine and plasma samples were assessed using assay kits from Wako Pure Chemical Industries. We calculated creatinine clearance (Ccr) by dividing the amount of creatinine excreted into urine samples by its plasma concentration. After an appropriate 4 dilution with 0.1% nitric acid, the concentrations of lithium in plasma (Li Cp) and urine were determined using the atomic absorption spectrometry Agilent 280Z AA (Agilent Technologies, Santa Clara, CA, USA). The renal clearance of lithium (Li CLr) was calculated by dividing the urinary excretion rate of lithium by its plasma concentration.
The fractional excretion of lithium was calculated by dividing Li CLr by Ccr, and its fractional reabsorption was evaluated by subtracting the ratio from one. The urinary ratio of lithium to creatinine was calculated by dividing the amounts of lithium by those of creatinine in urine samples.
Total RNA was extracted from the rat kidney cortex using TRIzol™ reagent (Thermo Data were expressed as mean ± SD, and were analyzed by unpaired t-test or one-way analysis of variance followed by Scheffé's test using KaleidaGraph (Synergy Software, Reading, PA, USA). Differences were considered to be significant at p < 0.05. Table 1 shows the pharmacokinetic parameters of lithium in rats infused with lithium chloride at 60 µg/min. Li Cp was significantly higher in female rats (1.66 ± 0.08 mEq/l) than in male rats (1.33 ± 0.10 mEq/l). Furthermore, Ccr and Li CLr were significantly lower, and the fractional reabsorption of lithium was significantly higher in female rats.
Urinary Li/creatinine was also significantly lower in female rats. These results suggest 5 superior lithium reabsorption in female rats at this plasma range of lithium. The plasma concentrations of lithium in rats infused with lithium chloride at 60 µg/min resemble those used in clinical settings. These sex differences in lithium reabsorption may also be observed under clinical conditions. Table 2 represents the pharmacokinetic parameters of lithium in rats infused with lithium chloride at 6 µg/min. No significant differences were observed in any of the parameters examined between the sexes in rats not infused with foscarnet. Plasma lithium concentrations in rats infused with lithium chloride at 60 and 6 µg/min were similar to those in rats that received lithium chloride injections at 25 and 2.5 mg/kg, respectively, as a bolus (Uwai et al. 2018(Uwai et al. , 2020. Fractional lithium reabsorption (excretion) in male rats infused with lithium chloride at 60 and 6 µg/min in the present study was similar to that by the low-affinity type and high-affinity type, respectively (Uwai et al. 2018(Uwai et al. , 2020. these results suggested that lithium reabsorption with low affinity was superior in female rats than in male rats.   Wistar/ST rats, suggesting that sodium-dependent phosphate transporter(s) mediate lithium reabsorption with high affinity (Uwai et al. 2014(Uwai et al. , 2018. At the proximal tubules, the sodium-dependent phosphate transporters NaPi-IIa, NaPi-IIc, and PiT-2 reabsorb phosphate filtered through the glomeruli (Biber et al. 2013). Since the mRNA expression level of PiT-2 was higher in female rats (Fig. 1) and the fractional reabsorption of lithium was similar in both sexes (Table 2), PiT-2 did not appear to significantly contribute to lithium reabsorption. It was accepted that the sodium-proton exchanger NHE3 should reabsorb lithium (Timmer and Sands 1999), and we assumed its role for the lithium reabsorption with the low affinity. Amiloride was previously shown to inhibit NHE3 as well as the epithelial sodium channel ENaC in the collecting duct, and its inhibitory effects on NHE3 were weaker than those on ENaC (Khadilkar et al. 2001;Yamamura et al. 2005). Fransen et al. (1992) reported that amiloride inhibited the reabsorption of sodium and lithium between the early distal tubules and urine, but not between the glomeruli and late proximal tubules in rats using a micropuncture experiment, suggesting the contribution of ENaC to lithium reabsorption. Therefore, the role of NHE3 in lithium reabsorption may be examined by administering amiloride at a markedly higher dose. A recent study demonstrated that NHE3 was not involved in lithium reabsorption using its knockout mice (Thomas et al.

2019)
, and no sex differences were observed in NHE3 mRNA expression levels.
Therefore, further studies are needed to identify which transporter(s) reabsorb lithium at the proximal tubules.
In conclusion, the present results demonstrated sexual dimorphism in the tubular reabsorption of lithium using Wistar/ST rats. Sex differences in the renal handling of lithium in patients need to be investigated in the future.