Enhanced ammonia secretion by proximal tubules from mice receiving NH4Cl: role of angiotensin II

Glenn T. Nagami
(With the Technical Assistance of Evelyn M. Warech)

Nephrology Section, Medical and Research Services, Veterans Affairs Greater Los Angeles Healthcare System at West Los Angeles, Los Angeles 90073; and School of Medicine, University of California, Los Angeles, California 90095


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Acidosis and angiotensin II (ANG II) stimulate ammonia production and transport by the proximal tubule. We examined the effect of short-term (18 h) in vivo acid loading with NH4Cl on ammonia production and secretion rates by mouse S2 proximal tubule segments microperfused in vitro with or without ANG II in the luminal microperfusion solution. S2 tubules from NH4Cl-treated mice displayed higher rates of luminal ammonia secretion compared with those from control mice. The adaptive increase in ammonia secretion in NH4Cl-treated mice was eliminated when losartan was coadministered in vivo with NH4Cl. Ammonia secretion rates from both NH4Cl-treated and control mice were largely inhibited by amiloride. Addition of ANG II to the microperfusion solution enhanced ammonia secretion and production rates to a greater extent in tubules from NH4Cl-treated mice compared with those from controls, and the stimulatory effects of ANG II were blocked by losartan. These results demonstrate that a short-term acid challenge induces an adaptive increase in ammonia secretion by the proximal tubule and suggest that ANG II plays an important role in the adaptive enhancement of ammonia secretion that is observed with short-term acid challenges.

transport; ammoniagenesis; acid-base physiology; losartan; ammonium chloride


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A MAJOR WAY THAT THE KIDNEYS defend the body against the introduction of excess acid is via the production and excretion of total ammonia (tNH3 = NH3 + NH<UP><SUB>4</SUB><SUP>+</SUP></UP>) (11, 19, 23, 27, 30). We have previously demonstrated (21) that chronic metabolic acidosis in vivo and acute metabolic acidosis in vitro stimulate tNH3 production by mouse S2 proximal tubule segments. Changes in tNH3 excretion occur rapidly in response to acid challenges even before the induction of new enzymes involved in renal ammoniagenesis (4, 29). No studies have examined the impact of short-term in vivo acid challenges on the production and secretion of tNH3 in isolated proximal tubule segments.

We previously demonstrated (21) that giving mice NH4Cl for 7 days induced adaptive increases in the rates of tNH3 production and secretion by mouse S2 proximal tubule segments. We and others also demonstrated (2, 15, 17) that angiotensin II (ANG II) has important effects on tNH3 production and secretion by the proximal tubule. Thus in addition to the known effects of ANG II on acid secretion, bicarbonate reclamation, and fluid reabsorption by the proximal tubule (5-8, 12, 13), ANG II enhances new bicarbonate generation via the production and secretion of tNH3.

Studies by Seikaly and colleagues (28) and Braam and co-workers (1, 22) demonstrated that ANG II is produced by the proximal tubule in such a manner that the concentrations of ANG II in the luminal and peritubular fluid compartments may be 100-1,000 times higher than in the systemic circulation. Concentrations of ANG II in the 10-9 M range may be present in the tubule fluid.

The purposes of the present studies were to 1) examine the effects of short-term (18-h) NH4Cl administration on renal tNH3 excretion and tNH3 production and secretion by mouse S2 proximal tubule segments, 2) determine the effects of blocking the type 1 angiotensin (AT1) receptor in vivo on the response of the kidney and proximal tubule to the short-term acid challenge, and 3) determine the direct effects of ANG II in vitro on tNH3 production and secretion by S2 segments from control and NH4Cl-treated mice. These studies demonstrated that 1) short-term acid administration stimulates the rates of renal tNH3 excretion and luminal tNH3 secretion by S2 proximal tubule segments without affecting tNH3 production rates; 2) the enhanced renal tNH3 excretion and tNH3 secretion rates by proximal tubules with short-term acid treatment are blocked when the AT1-receptor blocker losartan is coadministered with NH4Cl treatment; and 3) the stimulatory effect of in vitro ANG II on tNH3 production and secretion rates is greater in S2 segments derived from NH4Cl-treated mice compared with controls, and the effects of ANG II are inhibited by losartan in both controls and acid-treated mice.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. The studies performed were approved by the Animal Research Committee at the Veterans Affairs Greater Los Angeles Healthcare System. Male Swiss-Webster mice (Hilltop, Scottdale, PA), weighing 25-30 g, were maintained on Purina rodent chow. Mice were provided 0.3 M NH4Cl in 2% sucrose, 0.3 M NH4Cl in 2% sucrose with 100 mg/l losartan, 2% sucrose with losartan, or 2% sucrose alone (control) for 18 h (short term). At the end of 18 h, the mice were anesthetized with intramuscular injections of ketamine (0.09 mg/g body wt) and xylazine (0.01 mg/g body wt) and blood was obtained from the aorta for measurement of plasma total CO2 (tCO2) and potassium concentration. Urine was obtained from the bladder for determination of tNH3 and creatinine.

Microperfusion of mouse proximal tubule segments. S2 segments of the mouse proximal tubule comprising the late convoluted and early straight portions (0.9 ± 0.2 mm) were dissected from the outer cortical nephrons under direct microscopic visualization. Each S2 segment was placed in a temperature-regulated chamber mounted over the objective of an inverted microscope and was microperfused using concentric pipettes so that the luminal aspect of the S2 segment was cannulated and perfused with Krebs-Ringer bicarbonate (KRB) buffer (14, 18). The flow rates (20.4 ± 0.2 nl/min) did not differ among the groups studied. The S2 segment was bathed in ~300 µl of KRB buffer containing 0.5 mM L-glutamine pregassed with 95% O2-5% CO2 at pH 7.4 and 37°C.

Measurement of tNH3 production rates. In studies to determine tNH3 production rates by isolated perfused S2 segments, the bath solution was covered with pregassed mineral oil and continuously bubbled with a gas jet of 95% O2-5% CO2. The distal end of the perfused segment remained open to the bath medium so that tNH3 entered the bath solution via the fluid leaving the distal end of the perfused segment and via direct release into the bath medium through the basolateral aspect of the S2 segment. At the end of a 20- to 30-min incubation period, an aliquot of the bath solution was taken for analysis of tNH3 using a microenzymatic method that coupled the conversion of 2-oxoglutarate, NADH, and tNH3 to NAD+ and glutamate (18). The volume of the bath solution was determined from the degree of dilution of trypan blue dye added in known amounts to the bath solution at the completion of the study.

Measurement of luminal tNH3 secretion rates. In studies to examine luminal tNH3 secretion rates, the fluid leaving the distal end of the perfused segment was collected with a pipette (18, 20). Luminal tNH3 secretion rates equaled the rate at which tNH3 left the distal end of the perfused segment in timed luminal fluid collections.

Measurements of tCO2 and potassium concentrations. tCO2 was enzymatically determined from serum samples using the phosphoenolpyruvate carboxykinase reaction (Sigma). The measurements were linear over the range of concentrations observed. Potassium measurements were made by ion-sensitive electrode (16).

Solutions. KRB buffer solution contained the following electrolytes (in mM): 125 NaCl, 25 NaHCO3, 5 KCl, 1 MgCl2, 1 NaH2PO3, and 1 CaCl2. ANG II and losartan (Merck) were used in concentrations as specified in RESULTS. The low-sodium perfusion solution substituted N-methyl glucamine chloride for NaCl in the KRB buffer solution. All glutamine-containing solutions were freshly prepared using the purest form of L-glutamine available (Sigma).

Statistical analysis. Comparisons between two groups of data were done using Student's t-test, whereas comparisons among multiple groups were made using ANOVA with multiple comparisons by the method of Scheffé (26). All data are presented as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of losartan and short-term NH4Cl loading on serum tCO2, potassium concentration, and urinary tNH3 excretion. Groups of mice (n = 5/group) received the following in their drinking water for 18 h: 2% sucrose in water, 100 mg/l losartan in 2% sucrose, 0.3 M NH4Cl in 2% sucrose, or 0.3 M NH4Cl + 100 mg/l losartan in 2% sucrose (Table 1). Mice receiving losartan and sucrose in the drinking solution displayed no significant differences in serum tCO2 concentrations, potassium concentrations, or urinary tNH3 excretion per milligram of creatinine compared with control mice receiving sucrose alone. Mice receiving NH4Cl in the drinking solution displayed similar serum tCO2 and potassium concentrations as the controls but had higher urinary tNH3 excretion per milligram of creatinine. The lack of a decrease in the tCO2 concentration in mice receiving NH4Cl may have resulted from the short duration of the administration of NH4Cl and from the short-term compensatory effects of increased excretion of tNH3 that was observed. In contrast, mice receiving NH4Cl and losartan in the drinking solution displayed lower serum tCO2 concentrations and had lower rates of tNH3 excretion than mice receiving NH4Cl without losartan. The serum potassium concentrations did not significantly differ among the study groups. Thus although losartan by itself had no effect on renal tNH3 excretion, the enhanced renal tNH3 excretion that was observed with an 18-h NH4Cl challenge was blocked when losartan was coadministered with the NH4Cl treatment.

                              
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Table 1.   Effects of losartan and short-term NH4Cl challenge on ammonia excretion

In vitro microperfusion of mouse S2 proximal tubule segments. Under the in vitro microperfusion conditions employed, the tNH3 production and secretion rates were stable for >45 min after the onset of tubule incubation in vitro. All studies were completed within that time period.

Effects of NH4Cl administration in vivo on tNH3 production and net luminal tNH3 secretion by S2 proximal tubule segments perfused in vitro. In previous studies (21), we demonstrated that tNH3 production and secretion rates were enhanced in S2 proximal tubule segments derived from mice given NH4Cl for 7 days. In the present study, we examined the effect of an 18-h exposure to NH4Cl on the rates of tNH3 production and transport by isolated proximal tubule segments microperfused under normal bicarbonate and pH concentrations in vitro. The tNH3 production rates observed in S2 segments derived from mice receiving NH4Cl for 18 h were not significantly different from the rates observed in S2 segments derived from control mice not receiving NH4Cl. As depicted in Fig. 1A, the tNH3 production rates were not significantly different in S2 segments derived from NH4Cl-challenged mice vs. those from control mice (23.1 ± 1.3 vs. 20.7 ± 1.0 pmol · min-1 · mm-1; n = 5). By contrast, as shown in Fig. 1B, the rates of net luminal tNH3 secretion were higher in five S2 segments derived from NH4Cl-treated mice compared with five segments from controls (14.8 ± 0.3 vs. 10.1 ± 0.9 pmol · min-1 · mm-1; P < 0.05). When losartan was given with NH4Cl, the enhancement in luminal tNH3 excretion that was observed with NH4Cl loading alone was not seen (11.4 ± 0.4 pmol · min-1 ·mm-1; n = 5). When losartan was given without NH4Cl, the rate of luminal tNH3 secretion by S2 proximal tubules (10.2 ± 1.0 pmol · min-1 · mm-1; n = 5) did not significantly differ from the rate observed in S2 segments from control mice. Unlike the results obtained with a chronic (7-day) NH4Cl acid-challenge protocol (21), treatment with NH4Cl for 18 h failed to significantly increase tNH3 production rates in S2 segments. Nevertheless, the 18-h exposure to NH4Cl did result in an adaptive increase in luminal tNH3 secretion rates by S2 proximal tubule segments, and the administration of the AT1-receptor blocker losartan with the acid challenge blocked the adaptive increase in net luminal tNH3 secretion.


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Fig. 1.   Total ammonia production rates (A) by isolated S2 proximal tubule segments were not significantly affected by 18-h NH4Cl treatment with or without coadministered losartan. Luminal total ammonia secretion rates (B) were significantly higher in S2 proximal tubule segments derived from mice given NH4Cl without losartan compared with those from control non-NH4Cl-loaded mice and NH4Cl-loaded mice given losartan. *P < 0.05.

Effect of amiloride on luminal tNH3 secretion after 18-h acid challenge. We previously demonstrated (18) that microperfusing normal mouse S2 proximal tubule segments with the low-sodium perfusion solution containing 0.1 mM amiloride inhibits luminal fluid acidification by 100% and net luminal tNH3 secretion by 90%. These results were consistent with transport of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on the Na+/H+ exchanger (10). In the present study, we examined the effect of luminal perfusion with a low-sodium perfusate containing amiloride on net luminal tNH3 secretion by S2 proximal tubule segments derived from control mice and mice given NH4Cl for 18 h (Fig. 2). As with control S2 segments derived from non-acid-loaded mice, net tNH3 secretion was inhibited by >90% by perfusion with the low-sodium amiloride-containing solutions. Net luminal tNH3 secretion fell from 10.4 ± 0.8 to 1.0 ± 0.2 pmol · min-1 · mm-1 in S2 segments from control mice and from 15.1 ± 0.1 to 1.4 ± 0.4 pmol · min-1 · mm-1 in segments from acid-challenged mice (P < 0.01; n = 5/group). Thus the enhanced tNH3 transport mechanism observed in mice exposed to short-term acid loading appeared to be similar in nature to the mechanism observed in controls with both being inhibited by conditions that would inhibit Na+/H+ (NH<UP><SUB>4</SUB><SUP>+</SUP></UP>) exchange (10, 18).


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Fig. 2.   Luminal total ammonia secretion rates were markedly inhibited by the addition of amiloride to a low-sodium microperfusion solution in S2 proximal tubule segments derived from control mice and mice given NH4Cl. *P < 0.01 vs. without amiloride.

Effects of luminal ANG II on tNH3 production and net luminal secretion rates in S2 segments derived from NH4Cl-loaded mice. In our previous studies (17), we demonstrated that ANG II added to the luminal perfusion solution significantly stimulated tNH3 production in S2 segments derived from normal mice. In the present study, we examined the effects of 10-9 M ANG II in the tubule lumen on tNH3 production and net luminal secretion rates in control mice and mice given NH4Cl for 18 h before the study (n = 5/group). As depicted in Fig. 3, addition of ANG II to the luminal perfusion solution stimulated tNH3 production rates by S2 segments from both control mice (21.0 ± 0.6 pmol · min-1 · mm-1 without ANG II and 28.1 ± 1.0 pmol · min-1 · mm-1 with ANG II; P < 0.05) and acid-loaded mice (22.6 ± 0.9 vs. 39.4 ± 1.5 pmol · min-1 · mm-1; P < 0.01). ANG II had a significantly greater stimulatory effect on tNH3 production rates by S2 proximal tubule segments from NH4Cl-treated mice compared with its effects on S2 segments from nonacidotic controls (P < 0.05). In previous studies (17), we demonstrated that the stimulatory effect of ANG II on total ammonia production was blocked by concurrent provision of the angiotensin-receptor blocker saralasin to the luminal fluid. The stimulatory effect of luminal ANG II on the tNH3 production rate by S2 segments from acid-loaded mice was also inhibited by the addition of losartan (10-6 M) to the luminal fluid (21.4 ± 0.9 pmol · min-1 · mm-1). These studies indicate that acid loading increases the ammoniagenic response of the proximal tubule to luminal ANG II that is mediated through interactions with an AT1 receptor.


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Fig. 3.   Angiotensin II (ANG II; 1 nM) added to the lumen perfusion solution resulted in higher total ammonia production rates in S2 proximal tubule segments derived from control mice and mice given NH4Cl for 18 h. Total ammonia production rates were stimulated to a higher degree in S2 segments derived from mice that received NH4Cl. *P < 0.05 vs. others.

We also examined the effect of luminal 10-9 M ANG II on net luminal tNH3 secretion in S2 proximal tubule segments from control and acid-loaded mice. ANG II increased luminal tNH3 secretion by S2 proximal tubule segments from control mice (no ANG II, 11.1 ± 1.0 pmol · min-1 · mm-1 vs. ANG II, 20.0 ± 1.5 pmol · min-1 · mm-1; n = 5; P < 0.05; Fig. 4). The net luminal tNH3 secretion rate by S2 segments from mice receiving NH4Cl for 18 h was also markedly stimulated (no ANG II, 14.8 ± 0.5 pmol · min-1 · mm-1 vs. ANG II, 30.4 ± 1.6 pmol · min-1 · mm-1; n = 4; P < 0.01). The effect of ANG II on net luminal tNH3 secretion in S2 proximal tubule segments from NH4Cl-treated mice was greater than the effect observed in tubules from control mice (P < 0.05) and was blocked by 10-6 M losartan (15.3 ± 0.8 pmol · min-1 · mm-1; n = 5). Thus ANG II stimulates net luminal tNH3 secretion rates to a significantly higher extent in tubules from acid-loaded mice compared with those observed in non-acid-loaded controls. Furthermore, the effect of ANG II on luminal tNH3 secretion rates in NH4Cl-treated mice is abolished by the addition of losartan to the luminal perfusion solution.


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Fig. 4.   ANG II (1 nM) added to the lumen perfusion fluid resulted in higher luminal ammonia secretion rates by S2 segments compared with S2 segments microperfused without ANG II and resulted in higher total ammonia secretion rates in S2 segments from mice treated with NH4Cl compared with those derived from controls. *P < 0.05 vs. control without ANG II. **P < 0.05 vs. other groups.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In response to the introduction of excess acid to the body, the kidney increases urinary tNH3 excretion before the induction of enhanced amounts of enzymes involved in tNH3 production (4, 29). Thus the early response of the kidney to acid loading is to facilitate transport of tNH3 into the urine. Enhanced rates of transport occur in the proximal tubule in response to acute acid loading (29). We previously demonstrated that with prolonged (7-day) treatment with NH4Cl, both tNH3 production and secretion rates underwent adaptive enhancement (20). The results of the present study demonstrated that short-term (18-h) NH4Cl treatment induced an adaptive enhancement of luminal tNH3 secretion without inducing a significant adaptive increase in the tNH3 production rate. Thus the early response to acid challenges by the mouse proximal tubule may be to increase tNH3 transport mechanisms before inducing increased amounts of ammoniagenic enzymes. The secretion of tNH3 by the proximal tubule with its subsequent excretion into the urine would increase net acid excretion from the body.

ANG II has important effects on tNH3 production and transport in the proximal tubule (2, 15, 17). Luminal ANG II not only stimulates tNH3 production but also stimulates ammonia secretion into the luminal fluid (17). With acidosis in vivo the renin-angiotensin system has been shown to be upregulated (3, 9, 25). The results of the present study demonstrate that providing an inhibitor of AT1 receptors, losartan, with the NH4Cl challenge blocked both the increase in urinary tNH3 excretion and the adaptive increase in the net luminal tNH3 secretion rate by the proximal tubule that were observed with 18-h NH4Cl administration without losartan. The administration of losartan had no effect on the basal rates of urinary tNH3 excretion or the rates of tNH3 production or secretion by proximal tubule segments from control mice not treated with NH4Cl. These results suggest that ANG II plays an important role in modulating renal tNH3 excretion and tNH3 transport by the proximal tubule in response to acid challenges.

The major transport mechanism responsible for the increase in transport of tNH3 with NH4Cl treatment appeared to be fundamentally the same as the mechanism present under basal dietary conditions. As occurs in proximal tubules from control mice (18), luminal ammonia secretion in proximal tubules from NH4Cl-treated mice was substantially inhibited by perfusion of the luminal fluid with a low-sodium perfusate containing amiloride. Thus net luminal tNH3 secretion by S2 segments from control and acid-treated mice may be mediated via the Na+/H+ exchanger (18), and the enhanced transport rate observed in S2 segments from NH4Cl-treated mice may be due to the induction of additional amiloride-sensitive transporters or to the enhanced efficiency of existing tNH3 transporters.

Luminal ANG II markedly stimulated tNH3 production and secretion rates in proximal tubule segments dissected from non-acid-treated control mice and mice receiving NH4Cl treatment for 18 h. The increase in the tNH3 production rate in response to ANG II occurred in the absence of a significant rise in basal tNH3 production rates with NH4Cl treatment. A significantly greater increment in the luminal tNH3 secretion rate in response to the addition of ANG II to the luminal perfusion solution was observed in S2 segments from mice that received short-term acid loading in vivo compared with that observed in segments from controls. In other words, acid loading with NH4Cl in vivo greatly enhances the stimulatory effect of luminal ANG II in vitro on tNH3 production and secretory rates. The results of the present study differ from the results of Quan and Baum (24), who demonstrated that AT1 receptor and angiotensin-converting enzyme inhibition had a significant effect on fluid reabsorption in the absence of exogenous ANG II and that exogenous ANG II had no effect on fluid reabsorption rates. By contrast, our results demonstrated that the stimulation of tNH3 production and transport rates by exogenous ANG II was blocked by losartan, but the rates did not fall below basal values. The differences observed may be due to differences in species, experimental protocols, or the functions measured. The concentration of ANG II that was present in the fluid of the tubule lumen with in vitro microperfusion in our studies was unknown. It is likely that the luminal concentrations were much lower than concentrations reported with in vivo micropuncture sampling (1, 22, 28) due to dilution and the lack of upstream cells that could contribute to the final ANG II concentration. As a result, our studies do not rule out a possible paracrine role of ANG II in vivo. The ANG II that is present in the lumen of the proximal tubule in vivo could play a key role in enabling the proximal tubule to optimally generate and secrete more tNH3 to maintain acid-base balance in the face of acid challenges.

In a recent study in human subjects, Henger and colleagues (9) demonstrated the importance of ANG II in maintaining high renal tNH3 excretion rates in normal subjects with experimentally induced metabolic acidosis. They showed that in individuals with established NH4Cl-induced acidosis and high rates of tNH3 excretion, angiotensin receptor blockade led to net acid accumulation over a 4-day period that largely resulted from a reduction in urinary tNH3 excretion and led to a worsening of acidosis. Our experiments differed from those of Henger and co-workers in that the mice used in our studies were not initially acidotic when exposed to losartan and were not given an agent that blocks the action of aldosterone. In our study, the adaptive increases in tNH3 excretion rates by the kidney and tNH3 secretion rates by the proximal tubule in response to a short-term exposure to NH4Cl were blocked by concurrent administration of the AT1 receptor blocker losartan. Although the experimental design of the study by Henger and co-workers involved human subjects and pretreatment with NH4Cl and spironolactone, their results were complementary to ours: their results demonstrated the importance of ANG II in allowing the kidneys to maintain optimal tNH3 excretion with established acidosis, whereas our results demonstrated the role of ANG II in the enhancement of tNH3 excretion in response to a short-term acid challenge.

In summary, the present studies demonstrated that 1) short-term acid loading enhances urinary tNH3 excretion by mice and net luminal tNH3 secretion by isolated perfused mouse S2 segments derived from NH4Cl-treated mice, 2) ANG II plays an important role in the adaptive increase in urinary tNH3 excretion and S2 proximal tubule tNH3 secretion, and 3) short-term acid challenges increase the response of the proximal tubule to the stimulatory effects of ANG II on tNH3 production and luminal secretion. Taken together, these results indicate the important role of ANG II in the regulation of the adaptation and response of the proximal tubule to acid challenges.


    ACKNOWLEDGEMENTS

This work was supported by Medical Research Funds from the Department of Veterans Affairs.


    FOOTNOTES

Address for reprint requests and other correspondence: G. T. Nagami, Nephrology Section 111L, VA Greater Los Angeles Healthcare System at West Los Angeles, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail: gnagami{at}ucla.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.

10.1152/ajprenal.00249.2001

Received 9 August 2001; accepted in final form 15 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 282(3):F472-F477