Epithelial Na channels and short-term renal response to salt deprivation

Gustavo Frindt, Tiffany McNair, Anke Dahlmann, Emily Jacobs-Palmer, and Lawrence G. Palmer

Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York 10021


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To test the role of epithelial Na channels in the day-to-day regulation of renal Na excretion, rats were infused via osmotic minipumps with the Na channel blocker amiloride at rates that achieved drug concentrations of 2-5 µM in the lumen of the distal nephron. Daily Na excretion rates were unchanged, although amiloride-treated animals tended to excrete more Na in the afternoon and less in the late evening than controls. When the rats were given a low-Na diet, Na excretion rates were elevated in the amiloride-treated group within 4 h and remained higher than controls for at least 48 h. Adrenalectomized animals responded similarly to the low-Na diet. In contrast, rats infused with polythiazide at rates designed to inhibit NaCl transport in the distal tubule were able to conserve Na as well as did the controls. Injection of aldosterone (2 µg/100 g body wt) decreased Na excretion in control animals after a 1-h delay. This effect was largely abolished in amiloride-treated rats. On the basis of quantitative analysis of the results, we conclude that activation of amiloride-sensitive channels by mineralocorticoids accounts for 50-80% of the immediate natriuretic response of the kidney to a reduction in Na intake. Furthermore, the channels are necessary to achieve minimal rates of Na excretion during more chronic Na deprivation.

amiloride; polythiazide; aldosterone; adrenalectomy


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS GENERALLY RECOGNIZED that epithelial Na channels (ENaC) are important in the overall long-term regulation of Na balance and blood pressure. This is illustrated by disease states in which Na channels are defective. Mutations in the ENaC gene that reduce channel activity lead to the clinical syndrome of pseudohypoaldosteronism, characterized by salt wasting and hypotension (3, 30). Conversely, mutations in the beta - or gamma -ENaC genes that make the channels more active give rise to Liddle's syndrome, a monogenic form of hypertension in which Na is retained (and K is lost), despite low levels of mineralocorticoids (29, 34).

However, the precise role of aldosterone and Na channels in the day-to-day maintenance of Na balance has not been established. This is due, in large part, to the complexities of the regulatory systems involved in the control of plasma volume and blood pressure. First, changes in Na excretion (UNaV) can be brought about not only by changes in aldosterone levels but also by other hormones that affect the kidney. These include atrial natriuretic factor, the levels of which can decrease in response to volume contraction, and angiotensin II, which will vary in parallel with aldosterone and can affect the kidney directly (11). Second, there are multiple effects of these factors on the kidney through which UNaV can be altered. Atrial natriuretic factor can change glomerular filtration rate (GFR) and, perhaps, also decrease Na reabsorption in the inner medulla through inhibition of Na-Cl cotransport or nonselective cation channels (18). Angiotensin II can directly stimulate Na reabsorption in the proximal and distal nephrons (6, 33). Decreased blood pressure can enhance Na retention via sympathetic nerve signals or through direct effects on the kidney (11). Finally, aldosterone itself may have multiple mechanisms and sites of action. In addition to its effects on Na channels (24), it can induce higher levels of the thiazide-sensitive Na-Cl cotransporter in the distal convoluted tubule (16). It can also activate Na/H exchange in cultured renal cells through a nongenomic mechanism (20).

Several studies have demonstrated the activation of Na channels in the rat cortical collecting tubule (CCT) in response to chronic Na deprivation (24, 26, 31). Previously, we reported that reduction of Na intake over a period of only 15 h leads to a substantial activation of Na channels that could account for most of the observed increase in renal Na reabsorption (9). Here, we test this concept further by using a pharmacological "knockout" of the Na channels in the distal nephron. The results suggest two roles of Na channel activation: 1) they contribute to the immediate Na-conserving response, accounting for >= 50% of the initial fall in UNaV, and 2) they lower the minimal level of UNaV by ~10-fold.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Animals. Female Sprague-Dawley rats (120-180 g) raised free of viral infections (Charles River Laboratories, Kingston, NY) were fed a low-Na diet (3.8 mg Na/kg, 8.6 g K/kg; ICN, Cleveland, OH) or a matched control diet to which NaCl (10 g/kg) was added. Average consumption of food was 12 g/day for both diets. Na intake was ~2,100 meq/day on the control diet and essentially zero on the low-Na diet. Some animals were implanted subcutaneously with osmotic minipumps (model 2002, Alza, Palo Alto, CA) to deliver amiloride (Research Biochemicals, Natick, MA; 30 nmol/h) and/or polythiazide (PTZ; US Pharmacopeia, Rockville, MD; 15 nmol/h). The drugs were dissolved in polyethylene glycol 300 at concentrations calculated to give the desired infusion rate according to the pumping rate specified by the manufacturer. In some experiments, rats were injected subcutaneously with a single dose of d-aldosterone (2 µg/100 g body wt). To measure urinary excretion rates, animals were kept individually in metabolic cages (Nalge Nunc, Rochester, NY) with free access to food and water. Between 0800 and 1200, the rats were kept without food but with free access to drinking water lightly sweetened with 3% sucrose to increase water intake and urine flow. Adrenalectomized and sham-operated control rats were obtained from Charles River Laboratories.

Analytic methods. Rats were anesthetized with methoxyflurane, and blood was obtained from the abdominal aorta. Na and K were measured in plasma and urine by flame photometry (model 943, Instrumentation Laboratory, Lexington, MA). Urine Ca and Cl were measured colorimetrically with arsenazo III and mercuric thiocyanate, respectively, using commercial kits (Sigma Diagnostics, St. Louis, MO). Creatinine was measured with a commercial kit (Sigma Diagnostics) by a colorimetric method based on that described by Heinegard and Tiderstrom (13). Only female rats were used for creatinine clearance measurements, because creatinine is not secreted by the kidney in the female rat (12). Urine amiloride was measured fluorometrically according to Baer et al. (1).

Urine PTZ was analyzed by HPLC. The instrument (Waters, Milford, MA) included a system controller (model 600E), a photodiode array detector (model 996), and an injector (model U16K) and was operated under Millenium software. A reverse-phase C18 column (model 201TP54, Vydac) was used as the stationary phase; a concentration gradient of 20 mM ammonium acetate, pH 5.4 (solvent A), and acetonitrile (solvent B) was used as the mobile phase. The column was equilibrated at 100% solvent A before injection. After injection, solvent B was increased linearly to 20% over the first 2.5 min and then to 40% over the following 30 min. The flow rate was 1 ml/min. Under this gradient, PTZ had a retention time of 23.2 min and was well separated from other compounds present in urine in chromatograms obtained with the detector set at 269 nm. A calibration curve of peak area vs. PTZ over the range 100-1,000 ng was linear and was used to calculate the amount of the drug in urine.

Statistics. Statistical analysis of time course data was done by ANOVA or repeated-measures ANOVA, with post hoc comparisons made using the Tukey-Kramer test. In cases where UNaV decreased substantially, this method of analysis was insensitive to differences between groups that were large in relative terms but small in absolute terms. In these cases, we did two types of analysis. In the first, we used t-tests with correction for multiple comparisons. The P values required for significance were 0.05/n, where n is the number of comparisons in a given experiment. In the second method, we calculated means and standard deviations for the ratios of treated to control groups. We then performed ANOVA on these ratios and tested for significant differences with respect to baseline values, which were close to 1. Figures 1-9 report results of the first test, which was more stringent than the second.


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Fig. 1.   Amiloride infusion. A: time course of appearance of amiloride in urine. On day 0, rats were implanted with osmotic minipumps filled with amiloride. Amiloride concentrations were measured in urine collected from 0900 to 1200 over the next 3 days. Data are plotted as amiloride concentration (Uamil) and amiloride excretion rate (UamilV). B and C: effects of amiloride infusion on Na (UNaV) and K excretion (UKV), respectively. Differences in UNaV and UKV are significant only during the first 6 h of infusion. Values are means ± SE for 5 animals. **P < 0.01.



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Fig. 2.   Effect of amiloride infusion on response to an acute aldosterone injection. Rats were implanted with minipumps containing amiloride or sham operated (controls) 2 days before measurements began. Animals were placed in metabolic cages and fed a control-Na diet for 24 h. Urine was collected over this period, and average excretion rates were measured. Rats were then injected at 0900 with a single dose of aldosterone (2 µg/100 g body wt sc), and urine was collected for 3 subsequent periods of 1, 2, and 2 h.Results (means ± SE for 8 animals) are shown for UNaV (A), UKV (B), and Cl excretion UClV (C). D: time controls, in which animals received an injection of vehicle. Differences for individual time intervals were assessed by t-test with corrections for multiple comparisons. UNaV and UClV for control animals at 10-12 and 12-14 h were significantly different from baseline. Amiloride-treated animals were significantly different from controls at 1-3 h with respect to UNaV and UClV, but not UKV. **P < 0.01; ***P < 0.001.



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Fig. 3.   Effect of amiloride on circadian pattern of UNaV and UKV. Amiloride-treated and control animals were kept in metabolic cages and fed a normal-Na diet. Urine was collected every 4 h. Na (A) and K (B) were excreted in a circadian pattern. Excretion rates were highest during the night and early morning and lowest during the late morning and afternoon. The same general pattern was observed with amiloride-treated animals, but amplitude of variations was reduced in the case of UNaV, which was higher than in controls during the afternoon and lower during the night. Values are means ± SE for 6 animals. Significance assessed by ANOVA relative to peak: *P < 0.05; **P < 0.01; ***P < 0.001.



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Fig. 4.   Effect of amiloride on response to a low-Na diet. Excretion values were measured at 24-h baseline, and rats were given a low-Na diet at 1800. Urine was collected every 4 h until the following afternoon. UNaV was consistently higher in amiloride-treated animals (A), particularly during the first collection period (1800 to 2200) and in all time periods after 0600 the next morning. No significant effects on UKV were observed (B). Values are means ± SE for 6 animals. Overall effect of amiloride was highly significant (P = 0.003) on the basis of repeated-measures ANOVA. Differences for individual time intervals were assessed by t-test with corrections for multiple comparisons: **P < 0.01; ***P < 0.001. The last 3 time points were excluded from statistical analysis, because they include data for only 4 of 6 pairs.



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Fig. 5.   UNaV as a function of Na deficit. UNaV values are plotted on a logarithmic scale vs. mean Na deficit for the first 5 time periods of Fig. 4A.



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Fig. 6.   Effects of adrenalectomy on response to a low-Na diet. Amiloride-infused (A) or adrenalectomized (B) animals were fed a normal diet for 24 h (baseline) before being switched to the low-Na diet. In this simplified protocol, urine was collected overnight (1800 to 0900), during the late morning (0900 to 1200), and during the afternoon (1200 to 1800). UNaV was greater in amiloride-treated and adrenalectomized rats than in sham-operated controls. Effects of drug infusion and adrenalectomy were similar. Values are means ± SE for 6-9 animals. Differences for individual time intervals were assessed by t-test with corrections for multiple comparisons: **P < 0.01; ***P < 0.001.



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Fig. 7.   Polythiazide (PTZ) infusion. A: PTZ in urine. Rats were implanted with minipumps, and urine was collected over 6-h periods for 5 days. PTZ was measured using HPLC. Values are means ± SE for 5 animals. B and C: effects of PTZ infusion on UNaV and UKV. Differences in UNaV are significant only during the first 6 h of infusion. D: effects of PTZ infusion on urinary Ca excretion (UCaV). Urine was collected for a 3-h period before implantation of osmotic minipumps containing PTZ at time 0 (1200). Urine was then collected twice each day during the morning (0900 to 1200) and in the afternoon (1200 to 1800) for 3 days while animals were fed a control-Na diet and for an additional day while animals were fed a low-Na diet. Control animals excreted Ca with a strong circadian rhythm with high excretion rates during the morning and low rates during the afternoon. PTZ markedly inhibited UCaV during high-rate periods. Values are means ± SE for 5 animals. Differences for individual time intervals were assessed by t-test with corrections for multiple comparisons: **P < 0.01. Decrease in UCaV was significant on days 2 and 3 at 0900 to 1200 and combined 0900 to 1800 interval.



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Fig. 8.   Effects of PTZ infusion on UNaV. Urine was collected for 24 h while animals were maintained on a normal-Na diet and then for another 24 h on a low-Na diet. A: PTZ infusion did not alter pattern of Na excretion compared with sham-operated controls. B: PTZ did not alter pattern of Na excretion in amiloride-infused animals. Values are means ± SE for 7 (A) and 4 (B) pairs of animals. No significant effect of PTZ was observed.



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Fig. 9.   Effects of PTZ infusion on response to an acute injection of aldosterone. Procotols were similar to those shown in Fig. 2. UNaV fell to the same extent in PTZ-infused and control animals 1-3 h after a single injection of aldosterone (2 µg/100 g body wt sc). Values are means ± SE for 7 animals. No significant effect of PTZ was observed.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A major goal in this study was to develop an animal model in which renal Na channel function was abolished. Our approach was pharmacological. We aimed to infuse the Na channel blocker amiloride into the rat through osmotic minipumps at rates that would achieve steady-state levels in the urine sufficient to block channels in the distal nephron but to have minimal effects elsewhere. We chose the dose on the basis of the assumption that ~50% of the drug would be cleared from plasma through urinary excretion (1) by a combination of glomerular filtration and secretion by the proximal tubule. We further assumed that rates of fluid delivery to the channel-expressing parts of the distal nephron were ~60 µl/min, corresponding to 10% of GFR. Thus, when the infusion rate was 0.5 nmol/min, we estimated that 0.25 nmol/min would be excreted in a volume of 60 µl/min, giving a concentration of 4 µM in the distal tubular fluid.

Direct measurements of amiloride concentration in the urine confirmed that this estimate was reasonably correct. Figure 1 shows the time course of the rate of appearance of amiloride in the urine. The drug could be detected within 1 day after implantation of the minipump. The rate of excretion of amiloride in the urine reached a steady state after 2 days. One group of rats infused with amiloride and given drinking water sweetened with sucrose to promote drinking showed a marked water diuresis. Measurements of amiloride are shown in Table 1. The concentration in the final urine was ~5 µM. If it is assumed that little water was reabsorbed by the collecting duct under these conditions, this would also represent the approximate concentration in the lumen near the end of the CCT. If fluid delivery to the early part of the collecting duct system (i.e., the connecting tubule) is 10% of GFR or ~60 µl/min, the concentration at this point will be ~2.6 µM.

                              
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Table 1.   Amiloride measured in urine

If the inhibition constant (Ki) for amiloride is assumed to be 0.1 µM (25), 2-5 µM will block 95-98% of the Na channel activity in these segments. In contrast, if amiloride enters the urine primarily by filtration, the concentration in the glomerular filtrate (and, by inference, in plasma) will be ~0.25 µM. This would have minimal impact on Na reabsorption via the Na/H exchanger in the proximal tubule, inasmuch as the Ki for this transporter is ~50 µM (17). Furthermore, the thiazide-sensitive Na-Cl cotransporter is unaffected by amiloride at concentrations up to 100 µM (10). Thus, under these conditions, we believe that we can achieve a reasonably complete and a reasonably specific block of Na channels in the distal nephron in vivo.

Figure 1, B and C, shows the initial response of UNaV and K excretion (UKV) to amiloride infusion. There was a significant increase in UNaV and decrease in UKV during the first 6 h after implantation of the minipumps. After that time, the kidneys appeared to adapt to the presence of the drug, and excretion rates were similar to those in controls.

As a further test of the efficacy of blocking the channels, we examined the effects of an acute injection of aldosterone on UNaV. This hormone increases Na reabsorption, at least in part through increases in Na channel activity (28). Figure 2 shows the results of the injection in control and amiloride-treated animals. In controls, UNaV remained near basal levels for the first hour, reflecting the well-established lag time characterizing hormone action. In subsequent 2-h time periods, excretion rates dropped substantially as expected. Somewhat unexpectedly, UKV was not strongly affected by aldosterone with this protocol (Fig. 2B), while Cl excretion (UClV) fell in parallel with UNaV (Fig. 2C). We do not know why UKV was not increased under these conditions. The reductions in UNaV and UClV were significantly attenuated by amiloride. The difference in absolute values of UClV in control and amiloride-treated animals fell short of significance, but the differences in the fall in individual rats from baseline to the 10- to 12-h period were significant (P = 0.013). Even in the presence of amiloride, however, there was a significant decrease in UNaV with aldosterone injection. This may reflect a contribution from a hormone-dependent transporter other than the channels.

When urine was collected for 4-h periods over 24 h from control animals, a marked diurnal rhythm for UNaV and UKV emerged (Fig. 3). Peak rates of excretion for both ions occurred during the night and early morning, corresponding roughly to the highest rates of food intake. Lowest levels of excretion were in the late afternoon. Diurnal variations in UNaV were somewhat larger than those for UKV.

Figure 3 also shows the pattern obtained with amiloride-infused rats beginning 1 day after implantation of the minipumps. Over this period, the animals were in approximate Na and K balance, and Na intake and 24-h excretion rates were similar to those of controls. There was, however, a small but consistent change in the diurnal pattern of salt excretion. In the late afternoon (1400 to 1800), when UNaV reached its lowest levels, UNaV was higher in amiloride-treated rats than in controls. This pattern appeared to reverse during the night (0200 to 0600), during which UNaV was higher in controls. The ratio of UNaV at the lowest (1400 to 1800) to highest (0200 to 0600) time points was significantly lower in controls than in amiloride-treated animals (0.10 ± 0.02 vs. 0.47 ± 0.10, P = 0.014). Amiloride did not change the diurnal pattern of UKV significantly.

Figure 4 illustrates the time course of the fall in UNaV in response to a decrease in Na intake. A decrease in UNaV was evident even during the first 4 h of Na deprivation. This initial fall occurs from 1800 to 2200, a time when UNaV normally increases in parallel with increased food intake (Fig. 3). Intake was not significantly different from normal after the change in diet. Within 20 h, UNaV dropped to extremely low levels, representing <0.005% of the filtered load. UKV was not noticeably affected (Fig. 4B). Two effects of amiloride on the response to the low-Na diet are evident. The blocker slowed the initial fall in UNaV, and the largest absolute difference in excretion occurred during the first time interval of Na restriction. Amiloride also had a large effect on the minimal UNaV measured after 16 h. This rate, which persisted for at least the next 24 h, was ~0.08-0.2% of the filtered load, 20- to 50-fold higher than in controls. In the presence of amiloride, a small increase in UNaV during the second overnight period persisted, despite very low levels of Na intake.

The data from Fig. 4 are replotted in Fig. 5 as UNaV vs. Na deficit. These deficits were calculated taking Na intake as zero after the change to the low-Na diet. Two phases of the effects of amiloride are evident. First, the initial fall in UNaV in response to the deficit is reduced by ~80%. This suggests that a substantial fraction of increased Na reabsorption by the kidney during the initial response is mediated by Na channels. Second, the sharp drop in excretion, which in controls virtually eliminates UNaV and limits the deficit to ~200 µmol, does not occur. Instead, the animals continue to lose Na up to a cumulative deficit of at least 450 µmol, equivalent to ~3 ml of extracellular fluid. We did not continue the experiment beyond this point, but we suspect that the animals would eventually die of hypovolemic shock. In other experiments, we have maintained untreated rats on the low-Na diet for up to 2 wk with no major deleterious effects. Amiloride-sensitive Na channels appear to be essential for survival under these conditions.

We next investigated the possible mechanisms underlying the reduction in UNaV in the presence of amiloride. It is possible that, in the presence of amiloride, plasma volume decreases to such an extent that GFR is reduced. Reduced UNaV would then result from a decreased filtered load of Na, rather than from increased reabsorption. To test this theory, we measured creatinine clearance as an estimate of GFR. As shown in Table 2, amiloride had no measurable effect on GFR under conditions of Na restriction. In addition, Na restriction per se did not reduce GFR compared with animals under Na-replete conditions. Therefore, the reduction in UNaV over the first 24 h of a low-Na diet results from increased Na reabsorption in the absence and presence of amiloride.

                              
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Table 2.   Plasma electrolytes and GFR with different diets and drug treatments

Increased Na reabsorption in the presence of amiloride could be mediated by an aldosterone-upregulated transport system other than the Na channel. To test this idea, we assessed the reduction in UNaV in response to dietary Na restriction in adrenalectomized rats. For these and subsequent experiments, we used a simpler protocol in which just three urine collections were made: overnight (1800 to 0900), morning (0900 to 1200), and afternoon (1200 to 1800). The results using this protocol on the effects of amiloride were similar to those described above and are shown in Fig. 6A. The same protocol comparing control (sham-operated) and adrenalectomized animals is illustrated in Fig. 6B. The pattern of UNaV was very similar in the amiloride-treated and adrenalectomized rats. The finding that the adrenalectomized animals did not excrete more Na than the amiloride-treated group during the first 24 h of Na restriction supports the idea that the effects of aldosterone over this time period are largely dependent on activation of amiloride-sensitive Na channels. A caveat to this interpretation is that GFR was reduced in the Na-depleted adrenalectomized animals (Table 2). A reduction in filtered load could help compensate for diminished reabsorption through another transporter.

Another possibility is that other transporters are activated during Na depletion by aldosterone-independent mechanisms. One candidate is the Na-Cl cotransporter, which is expressed in the distal convoluted tubule of the rat kidney (21) and is therefore in a good position to regulate the final composition of urine. We employed a similar strategy to investigate the possible role of this transporter. The potent thiazide diuretic PTZ was infused via osmotic minipumps at 250 pmol/min. PTZ measured using HPLC was 1-10 µM in the urine at the time of reduction of dietary Na, and the excretion of PTZ was highest just after implantation (70 pmol/min), declining to ~30 pmol/min thereafter (Fig. 7A). Therefore, in the steady state, ~10% of the drug is excreted intact in the urine. If it is assumed that GFR is 0.7 ml/min and that 10% of the filtered fluid is delivered to the distal convoluted tubule, we estimate that the concentration of the drug at its main site of action was 0.4 ± 0.1 µM at the time of reduction of Na intake. This is at least eight times the Ki of <0.05 µM for PTZ's effects on the cloned Na-Cl cotransporter under physiological conditions (19). This indicates that the transporter should be mostly inhibited under these conditions.

Two physiological measurements confirmed the effect of PTZs. First, during the first 6 h after implantation of the minipumps, UNaV was higher than in controls (Fig. 7B). UKV tended to increased during this period, but the effect was not statistically significant (Fig. 7C). After that time, the excretion rates were similar in PTZ-treated and control groups, suggesting that the kidneys had adapted to inhibition of the cotransporter. Similar results were reported previously (7). Another hallmark of the action of the thiazides on the distal tubule is a reduction in urinary Ca excretion (UCaV). Figure 7D shows the effects of PTZ infusion on UCaV after implantation of the minipumps. In untreated rats, there was a strong diurnal rhythm of UCaV, with rates much higher in the morning than in the afternoon. PTZ greatly reduced the morning UCaV and strongly blunted the diurnal variations. Although the mechanism by which thiazides stimulate Ca absorption is controversial, the effects are thought to be secondary to inhibition of the Na-Cl cotransporter (8). Thus three observations indicate that drug concentrations high enough to block the cotransporter are achieved by this protocol.

The effects of PTZ on the response to a low-Na diet are shown in Fig. 8A. UNaV values in the controls were not significantly different from those in the PTZ-treated group. After 18 h, UNaV levels were reduced to <0.01% of filtered load. This indicates that the Na-Cl cotransporter is not essential in the short-term response to reduced Na intake. It is possible that the cotransporter becomes important when the channels are blocked. To test this theory, we compared rats treated with amiloride + PTZ with those treated with amiloride alone (Fig. 8B). Again, UNaV was substantially higher than in controls (cf. Fig. 8A), but PTZ + amiloride did not have a marked effect on the excretion pattern.

We also tested whether the Na-Cl cotransporter might be involved in the acute response to aldosterone, as shown in Fig. 2. A similar protocol was carried out using control and PTZ-infused animals. There was no effect of thiazide on the response to aldosterone injection (Fig. 9). In this set of experiments, UNaV in both groups tended to increase toward basal levels 3-5 h after the injection. The results are consistent with a minimal role of the Na-Cl cotransporter in the acute effects of aldosterone on renal Na transport.

The effects of the various conditions on plasma electrolytes and GFR are shown in Table 2. As we reported previously (7), plasma K increased after 18 h of Na restriction. This hyperkalemia was even greater in the amiloride-treated and adrenalectomized groups, whereas the PTZ-treated animals were hypokalemic. Of the protocols used, only adrenalectomy had a significant impact on GFR.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pharmacological elimination of Na channels. The pharmacological blockade of Na channel activity has several advantages over the genetic knockout approach for addressing the questions posed in this study. Most importantly, the administration of the channel blocker amiloride was limited to a few days, rather than the lifetime of the animal, avoiding problems of respiratory failure, which can be lethal in alpha -ENaC-knockout mice (15), as well as possible developmental changes and adaptations to the missing transport activity. We also took advantage of the fact that the drug is cleared from the body largely by excretion in the urine, so that it was naturally concentrated in the lumen of the distal nephron, exactly the place where we wanted it to act. Finally, the technique could be conveniently used in the rat, an animal in which a great deal of information about renal function, in general, and Na channel activity, in particular, is known.

A major disadvantage of the pharmacological approach is that the drugs have limited specificities. Of particular concern in our study was the finding that amiloride can inhibit a variety of Na transport processes in addition to the Na channels, including Na/H and Na/Ca exchange (2, 17). Much larger doses are needed to affect these transporters, however, and concentrations in the low micromolar range will block most of the channel activity (Ki ~ 10-7 M) with minimal effects on the exchangers (Ki ~ 10-4 M). As mentioned above, the tendency for amiloride to be concentrated in the urine in the distal nephron, where the channels are located, relative to the proximal tubular fluid, where the Na/H exchanger mediates Na reabsorption, will augment the effective specificity of the drug for the channels.

The kidney can adapt to treatment with diuretics. Although baseline UNaV levels (Figs. 2, 4, 6, and 8) were similar in control and amiloride- or thiazide-treated animals, in the latter groups loss of one transporter activity may be compensated for by upregulation of others. This adaptation probably explains why UNaV was increased during the first 6 h of thiazide infusion, but not at later times.

Diurnal patterns of excretion. The diurnal rhythm of UNaV has been reported previously. Holtzman et al. (14) found that UNaV in the rat fell to a low basal level in the late morning after food intake diminishes. The diurnal variation was reduced when intake was spread out over the day. In our study, UKV also exhibited a diurnal pattern that was less marked than that for UNaV. The smaller variation in K output may reflect the ability of the intracellular compartment to buffer changes in extracellular K resulting from K intake.

Changes in Na channel activity seem to account in part for the diurnal pattern. Wang et al. (32) reported that urinary Na-to-K ratios in the mouse were higher in the morning than in the afternoon and that these changes were well correlated with the amiloride-sensitive potential difference across the distal colon, measured in vivo. A similar diurnal pattern was reported for the short-circuit current and the density of amiloride-sensitive Na channels in the rabbit colon, measured in vitro (5). In agreement with these results, we found that amiloride-infused animals excreted more Na than did controls in the late afternoon, when excretion rates were lowest. This suggests that channel-mediated Na reabsorption contributes to a reduction in UNaV during this time period. However, the diurnal rhythm, although blunted, persists in the amiloride-infused animals, indicating that other processes are also involved.

Increased channel activity in the late afternoon may be driven by higher levels of circulating aldosterone (5, 32). These changes could reflect differences in the rate of salt intake as food consumption decreased markedly during the day. However, a small nocturnal natriuresis was observed in amiloride-treated animals, even when the diet was Na free (Fig. 4A). This suggests that part of the increased UNaV during the night is driven by an inherent circadian rhythm or by the intake of food in general, rather than Na specifically.

Na channels in the response to low Na. We previously found that Na channels in the rat CCT were activated after 15-18 h of dietary Na restriction (9). A quantitative assessment suggested that the channels could account for a substantial fraction of the measured increase in Na reabsorption observed during this time interval in the Na-depleted state. This conclusion, however, was based on the assumption that the channels in the CCT in vivo could conduct Na at essentially maximal rates. This might not be true if, for example, Na delivery to the distal nephron were diminished and the luminal Na concentration fell below saturation levels. The main goal of this study was to use an independent test to evaluate this possibility. We used the amiloride-infused rat model to examine whether blocking the channels did indeed increase UNaV under these conditions and, if so, to what extent.

It is clear that without the channels the ability of the kidney to conserve Na was reduced. The effects of blocking the channels were observed in the first few hours after Na intake was reduced, when the largest absolute amiloride-sensitive Na reabsorption occurred. It is also evident that UNaV is reduced, even in the presumed absence of functional channels. A more quantitative conclusion is more difficult to reach, because, in the presence of the inhibitor, more Na is lost over the same time period, leading to a greater Na deficit and perhaps to the recruitment of other salt-conserving processes. One method of quantifying the role of the channels is therefore to compare the effect of amiloride on UNaV at similar degrees of Na depletion. This type of plot is shown in Fig. 5. The initial negative slope of the relationship of excretion vs. Na deficit is reduced by ~80% in the presence of amiloride. This suggests that a large fraction (>50%) of the increased Na reabsorption in response to a reduction in Na intake is mediated by the Na channels. This supports the conclusions based on direct measurements of channel activity.

A similarly large fraction of the response to an acute injection of aldosterone was abolished in the amiloride-infused animals. This finding, together with the observation that adrenalectomy and amiloride treatment affected UNaV to a similar extent, is consistent with the involvement of mineralocorticoids in the early Na-conserving process.

The effects of amiloride on UNaV after longer periods (>16 h) of Na deprivation were perhaps even more dramatic. In control animals, Na reabsorption increased markedly as the cumulative Na deficit approached ~200 µmol. After 16 h of salt restriction, UNaV virtually ceased and the animals survived the nearly complete absence of dietary Na for long periods of time. This ability to adapt to very low levels of salt intake is severely compromised when Na channels are blocked, and the animals continue to lose Na at significant rates. We conclude that the channels are essential to keep Na losses below ~0.1% of the filtered load.

Na-Cl cotransporters in the response to low Na. Under normal conditions, the thiazide-sensitive Na-Cl cotransporter in the distal convoluted tubule is thought to reabsorb a large fraction of the filtered load (27). In addition, chronic Na depletion increased levels of cotransporter protein, indicating a role at least in long-term Na conservation. Administration of PTZ, a potent thiazide diuretic, at levels sufficient to block the cotransporter, resulted in a transient increase in UNaV and a chronic decrease in UCaV. During short-term Na deprivation, the kidneys were able to reduce UNaV normally, despite the presence of the blocker. This result should be interpreted with caution, since the kidney can adapt to thiazide administration by upregulating levels of the cotransporter itself (4) as well as those of the Na channels (22). We do not eliminate the possibility that the cotransporter is involved in the short-term response to Na deprivation. However, it does not appear to be as essential as the Na channels in reducing UNaV to minimal levels.

Other transport systems involved in acute regulation of UNaV. Even in the presence of amiloride, the kidney can reduce UNaV substantially, from ~1-2% of filtered load under control conditions to ~0.1% with low Na intake. We do not know the processes that are involved in this response or whether they take place even when Na channels are active. GFR is not significantly changed after 15 h of low Na, even in the presence of amiloride (Table 2). Thus the decrease in excretion depends on an increase in Na reabsorption. As discussed above, the thiazide-sensitive cotransporter is not essential for this additional reabsorption, although it may contribute to it under normal conditions. A more likely candidate would be the Na/H exchanger in the proximal tubule. This exchanger is known to be stimulated by angiotensin II, and levels of this hormone presumably rise during Na depletion. A genetic knockout of the renal angiotensin II receptor does indeed interfere with Na conservation by the mouse kidney, despite normal increases in plasma aldosterone (23). It is not known to what extent this effect involves changes in tubular transport or in renal hemodynamics. If angiotensin II-dependent Na reabsorption in the proximal tubule were activated during Na restriction, delivery of Na to the distal nephron would be reduced, accounting for the low rates of UNaV, even in the absence of functioning Na channels.


    ACKNOWLEDGEMENTS

We thank Drs. Y. Q. Chen and J. Buck for making the HPLC apparatus available and for guidance in its use.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-11489 and DK-59659 to the Gateways to the Laboratory Program of Weill Medical College.

Address for reprint requests and other correspondence: L. G. Palmer, Dept. of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021 (E-mail: lgpalm{at}med.cornell.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

April 23, 2002;10.1152/ajprenal.00379.2001

Received 28 December 2001; accepted in final form 16 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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