Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The amiloride-sensitive epithelial Na⁺ channel is found in tight epithelia from frog skin, toad urinary bladder, turtle colon, and mammalian cortical-collecting tubules. It is characterized not only by its sensitivity to amiloride, but by its small conductance, its high selectivity for Li⁺ and Na⁺, and its slow kinetics (Benos, 1982; Palmer and Frindt, 1988). Although the channel exhibits a permeability for Li⁺ that is at least as high as for Na⁺ (Palmer and Frindt, 1988), Li⁺ is pumped at a slower rate through tight epithelia such as frog skin (Leblanc, 1972; Laski and Kurtzman, 1983) because Li⁺ is only a mediocre activator of Na-K-ATPase (Skou, 1960; Gutman et al., 1973; Siegel et al., 1975; Rodland and Dunham, 1980; Diamond et al., 1983; Halm and Dawson, 1983) and therefore accumulates intracellularly (Leblanc, 1972).

It has been demonstrated by in vitro studies in toad and turtle urinary bladder that Li⁺ inhibits the transport of Na⁺ by some unknown mechanism that is related to the intracellular Li⁺ concentration (Singer et al., 1972; Arruda et al., 1980; Bank et al., 1982; Herrera et al., 1985). Although it is known from studies in mammals that Li⁺ treatment leads to renal Na⁺ loss (Radomski et al., 1950; Baer et al., 1970, 1973; Thomsen, 1973, 1976; Thomsen et al., 1974), the mechanism has yet to be determined. Li⁺-treated dogs and rats have a reduced renal response to aldosterone or deoxycorticosterone acetate (DOCA) (Radomski et al., 1950; Schou, 1958; Baer et al., 1973; Thomsen et al., 1976; Iaina et al., 1982), and micropuncture studies in anesthetized animals have shown that acute administration of high-dose Li⁺ can inhibit Na⁺ reabsorption in the distal tubule (Galla et al., 1975; Hecht et al., 1978). However, the possible involvement of the amiloride-sensitive Na⁺ channels of principal cells has not been investigated.

The aim of the present study was to examine more directly whether the transport of Na⁺ through the amiloride-sensitive Na⁺ channel of the distal nephron is, in fact, inhibited by Li⁺ treatment. If it is, the channel should respond to neither aldosterone nor amiloride. The experiments were performed using a newly developed technique which permits studies in conscious, unstressed rats with servo-controlled maintenance of Na⁺ and fluid balance (Spannow et al., 1997). Thus, alterations in Na⁺ balance induced by aldosterone or amiloride treatment, which might have hampered interpretation of the findings, were avoided.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals and Physical Environment. Specific pathogen-free female Wistar rats weighing 200 to 260 g were used. The animals were housed in a temperature- (22-24°C) and moisture (60%)-controlled room with a 12-h light/12-h dark cycle. The rats were fed a wet-mash diet containing 500 mmol of Na⁺ and 200 mmol of K⁺ per kg dry weight for at least 4 weeks before experimentation. Half of the rats in addition were given Li⁺ in the food (100 mmol/kg dry weight) for 3 to 4 weeks immediately before experimentation. All rats were given free access to tap water, and the lithium-treated rats in addition were given free access to 0.46 M NaCl solution to compensate for the Li⁺-induced renal loss of Na⁺ (Jensen et al., 1976).

Surgical Preparation. Ten to 20 days before experimentation, the animals were anesthetized with halothane/N₂O. Using aseptic surgical techniques, sterile Tygon catheters were advanced into the abdominal aorta and the inferior caval vein via the femoral vessels, and a sterile chronic suprapubic bladder catheter was implanted into the bladder. All catheters were produced and fixed, with small modifications, as described by Petersen et al. (1991). After instrumentation, the rats were infused with saline (5 ml), given a long-lasting analgesic (Temgesic; Reckitt & Coleman, Hull, UK 10 µg/animal, s.c.), and allowed to recover from anesthesia. The arterial and venous catheters were sealed with 50% glucose solution containing 500 U of heparin and 10,000 U of strepkinase per ml. After the operation the rats were returned to the animal unit and housed individually. After a recovery period of 5 to 6 days, the rats were acclimatized to restriction by three daily training sessions in the restraining cages. The duration of each daily session was increased stepwise from 1 to 3 h a day.

Experimental Groups. Within each group (lithium-treated or control), the animals were allocated randomly to one of three experimental procedures: 1) infusion of vehicle alone throughout the experiment, 2) infusion of aldosterone throughout the experiment, or 3) infusion of aldosterone throughout the experiment, together with amiloride for the final 90 min.

Clearance Protocol. Each experiment comprised a 15-min bolus period for [³H]inulin and aldosterone (where applicable), a 120-min equilibration period, three 20-min urine collection periods, an 8-min bolus period for amiloride (where applicable), followed by a 22-min equilibration period and by three 20-min urine collection periods during which amiloride or vehicle was infused.

Maintenance of Na⁺ Electrode. The Na⁺ electrode was calibrated with standard solutions containing 10, 50, and 100 mM NaCl solution in 5 mM KCl solution. After each experiment, the electrode was conditioned with "Na/pH solution" (Novabiochem)and then perfused with 10 mM NaCl solution in 5 mM KCl solution until the next experiment while the Na⁺ concentration was measured continuously and data were collected. In this way, the stability and readings of the electrode could be checked by one glimpse at the computer screen before the start of the experiment and, if necessary, the calibration adjusted. After the experiment, the computer-calculated Na⁺ excretion was compared with the Na⁺ excretion based on measurements of urinary Na⁺ by flame-emission photometry. In every case, there was a good agreement between the two values.

Analysis. Urine volume was determined gravimetrically. Concentrations of Na⁺ and potassium in plasma and urine and Li⁺ in plasma were determined by flame-emission photometry. [³H]Inulin concentrations in plasma and urine were determined by liquid scintillation counting on a Packard Tri-Carb liquid scintillation analyzer. Fifteen microliters of the sample and 285 µl of water were mixed with 2.5 ml of Ultima Gold (Packard Instruments, Meriden, CT).

Calculations. Renal clearances (C) and fractional excretions (FE) were calculated by the standard formulas:
where U is the urine concentration, V is the urine flow rate, P is the plasma concentration, and GFR is the glomerular filtration rate as measured by [³H]inulin clearance.

Data Presentation and Statistics. All values are presented as means ± S.E.M. The values for renal clearance variables are derived from the averages of the three preamiloride periods and of the three periods during amiloride infusion. Overall statistical comparisons were performed by one-way ANOVA (between groups), one-way ANOVA for repeated measures (within group), or two-way ANOVA for repeated measures for two-way classified data (group and time). Individual comparisons within or between groups were performed by subsequent use of Student's paired or unpaired t test. Differences were considered statistically significant at the 0.05 level.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Renal Na⁺ excretion was measured continuously during the experiments by the Na⁺ electrode (Fig. 1). In the control rats, Na⁺ excretion decreased significantly in the time interval 0 to 195 min in each of the two subgroups given aldosterone when compared with the subgroup given vehicle alone. After blockade of the aldosterone-stimulated Na⁺ reabsorption by amiloride, Na⁺ excretion rose, whereas it remained unaltered in the two subgroups not given amiloride. In the Li⁺-treated rats, Na⁺ excretion from the beginning was higher than that of the control rats, confirming the natriuretic effect of Li⁺ treatment, and for all three subgroups taken together there was a small increase of Na⁺ excretion during the study. However, neither aldosterone nor amiloride affected Na⁺ excretion significantly.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Na+ excretion in control and Li+-treated rats. Within each of these groups, one subgroup was given vehicle only, the second was given aldosterone alone throughout the study, and the third was given aldosterone throughout the study, with amiloride superimposed during the time interval 195 to 285 min. Error bars represent S.E.M. for last urine collection period. Aldosterone versus vehicle for time interval 0 to 195 min: *p < .05, **p < .01; amiloride + aldosterone versus aldosterone alone for time interval 195 to 285 min; p < .001 (two-way ANOVA, group by time).

Mean arterial blood pressure (MAP) and plasma electrolytes before and during amiloride infusion are given in Table 1. The blood pressure was significantly higher in the lithium-treated group than in the control group. There were no significant differences in plasma Na⁺ or K⁺ between the two groups. Administration of amiloride influenced neither MAP nor the plasma electrolyte concentrations.

Renal clearance data are shown in Table 2. In the rats given vehicle only, GFR was significantly higher in the lithium-treated rats than in the control animals. C_Na and FE_Na also tended to be higher in the Li⁺-treated rats, although statistical significance was not quite reached. In the control animals, there was a tendency to a reduction in C_Na and FE_Na in response to aldosterone (compared with vehicle) and a significant increase in C_Na and FE_Na in response to amiloride. In the lithium-treated group, aldosterone had no significant effect on C_Na and FE_Na; furthermore, although there was some increase of C_Na and FE_Na in response to amiloride, the increase was less pronounced than that observed in the control group, and in any case some increase (although statistically insignificant) was also observed in the two time-control lithium-treated subgroups not given amiloride. Moreover, in lithium-treated rats, neither C_Na nor FE_Na was significantly higher in the aldosterone-amiloride subgroup during period 4 + 5 + 6 than in the aldosterone alone subgroup during the corresponding period.

After blockade of Na⁺ reabsorption in the collecting ducts by amiloride, FE_Na was very similar in the control group and the lithium-treated group (3.45 ± 0.74% versus 3.46 ± 0.33%), suggesting that the load of Na⁺ to the amiloride-sensitive site was similar in the two groups. In contrast, in the same time period there was a marked difference in FE_Na between the two groups given aldosterone alone (0.72 ± 0.32% versus 2.54 ± 0.55%, p < .05), reflecting a difference in Na⁺ reabsorption through the amiloride-sensitive channels. Fractional potassium excretion was not significantly different between the two groups before administration of amiloride and decreased to similar values after the administration of amiloride.

Further information about the amiloride-sensitive Na⁺ reabsorption appears from inspection of results from individual rats (Fig. 2). In control rats, the amiloride-sensitive Na⁺ reabsorption ( FE_Na) rose linearly with increasing load of Na⁺ to the amiloride-sensitive site (FE_Na-amil), at least when the latter did not exceed 5%. In one extreme data set (not shown), FE_Na-amil and  FE_Na were 9.2 and 6.2%, respectively, suggesting that when the delivered load is very high the capacity for Na⁺ reabsorption may be exceeded. In marked contrast to the control group,  FE_Na in the Li⁺-treated group remained at the same low level of approximately 1% in all rats independently of the Na⁺ load.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Amiloride-sensitive Na+ reabsorption ( FENa) as a function of the fractional Na+ load to the amiloride-sensitive site (FENa-amil) in control and Li+-treated rats.  FENa is calculated as difference between values for FENa pre- and postamiloride administration. Lines are regression line for control rats ± 2 × S.D. for residuals.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The essential findings of the present study in Li⁺-treated rats were 1) the absence of a response to aldosterone and 2) a much reduced response to amiloride. However, the main focus was not to investigate the effect of aldosterone per se, but to use aldosterone to prime the Na⁺ channels in the distal nephron in order to test the effect of amiloride, which has not been reported previously. The results show unequivocally that amiloride-sensitive Na⁺ transport is greatly reduced in Li⁺-treated rats.

Amiloride blocks conductive apical Na⁺ channels in principal cells in the collecting duct and is used as a tool to recognize Na⁺ reabsorption through these channels (Benos, 1982). The dose employed in the present study leads to intratubular amiloride concentrations well below those reported to block the Na⁺-H⁺ exchange mechanism in the proximal tubules but above the concentrations required to block movement of Na⁺ through the conductive Na⁺ channel (Shalmi et al., 1998). In line with this, micropuncture studies have demonstrated that Na⁺ reabsorption in the proximal tubule, the loop of Henle, or the distal tubule is not influenced and that the increased fractional urinary Na⁺ excretion is entirely a result of inhibition of Na⁺ reabsorption in the collecting duct (Fransen et al., 1992; Shirley et al., 1992; Walter et al., 1995). An increased urine flow rate during amiloride administration, as observed in the present study, has been found consistently and is due to decreased water reabsorption in the collecting ducts (Shirley et al., 1992).

As explained in Materials and Methods, assuming that amiloride completely blocked the conductive Na⁺ channels in the collecting ducts and that Na⁺ is not reabsorbed by other mechanisms in this segment (Shirley et al., 1992), the load of Na⁺ delivered to the amiloride-sensitive site can be estimated as the urinary excretion of Na⁺ after administration of amiloride (FE_Na-amil). That FE_Na-amil in control and Li⁺-treated rats was almost identical suggests that the load of Na⁺ to the amiloride-sensitive site was similar in the two groups and that the marked difference in Na⁺ excretion in the absence of amiloride was a result of a difference in Na⁺ reabsorption through the amiloride-sensitive channels in the distal nephron. Furthermore, in control rats, amiloride-sensitive Na⁺ reabsorption was found to increase with increasing Na⁺ load, as shown by the close correlation between  FE_Na and FE_Na-amil (Fig. 2). In contrast, in Li⁺-treated rats,  FE_Na did not rise with increasing Na⁺ load. Thus, these findings reaffirm that chronic Li⁺ treatment is associated with a reduction in amiloride-sensitive Na⁺ reabsorption. Although micropuncture studies in anesthetized animals have indicated that high doses of Li⁺ can inhibit Na⁺ reabsorption in additional nephron segments including proximal and distal convoluted tubules (Martinez-Maldonado et al., 1975; Hecht et al., 1978), the inhibitory site of action is likely to depend critically on the plasma Li⁺ concentration (very high in the studies cited) and on the duration of treatment (Thomsen, 1973). It is clear from the present investigation in conscious animals that the natriuretic effect of moderate plasma Li⁺ concentrations (~0.5 mM) is entirely due to inhibition of Na⁺ reabsorption at the amiloride-sensitive site.

Previous studies showing that Li⁺ inhibits Na⁺ transport through the amiloride-sensitive Na⁺ channel in toad urinary bladder (Singer et al., 1972; Herrera et al., 1985) and turtle bladder (Arruda et al., 1980; Bank et al., 1982) are consistent with the findings of the present investigation. Further evidence for inhibition of the amiloride-sensitive Na⁺ channel comes from studies showing a blunted renal response to DOCA or aldosterone in Li⁺-treated animals (Radomski et al., 1950; Schou, 1958; Baer et al., 1973; Iaina et al., 1982). However, those studies were carried out in animals with intact adrenal glands and, therefore, do not exclude the possibility that the blunted response could be a result of an increased level of endogenous aldosterone secretion secondary to lithium-induced sodium losses; high circulating levels of endogenous aldosterone would be expected to prevent any further effect of exogenous aldosterone. Thomsen et al. (1976) attempted to overcome this problem by using adrenalectomized rats. They used the voluntary intake of hypertonic NaCl solution as an index of renal Na⁺ losses and found that the reduction in saline consumption, which occurs in response to DOCA or aldosterone, was blunted in Li⁺-treated animals. In this context, it is worth noting that Stewart et al. (1987) described a patient with primary adrenal failure requiring gluco- and mineralocorticoid replacement therapy who developed mineralocorticoid resistance when taking Li⁺ for a manic-depressive illness. It should be re-emphasized, however, that no previous study has investigated amiloride-sensitive renal Na⁺ transport during Li⁺ treatment directly by the administration of amiloride.

The mechanism by which lithium inhibits the amiloride-sensitive Na⁺ channel is unknown. However, it is well known that lithium inhibits the Na⁺/H⁺ antiporter in many tissues including that of tight epithelia such as rat inner medullary collecting duct (Wall et al., 1988). Because the Na⁺/H⁺ antiporter is of vital importance for regulating the intracellular pH of the collecting duct cells (as in most other cells) (Preisig and Alpern, 1991), intracellular pH is likely to be reduced. Several studies have shown that a reduction in intracellular pH leads to a dramatic reduction of the apical Na⁺ conductive transport in tight epithelia including rat cortical-collecting ducts (Palmer and Frindt, 1987; Lyall et al., 1995).

In conclusion, the present results indicate that long-term lithium treatment, even when plasma Li⁺ concentrations are kept below 1 mM, reduces aldosterone-stimulated Na⁺ transport through the amiloride-sensitive Na⁺ channels in the distal nephron.